How to prepare culture media for plant tissue culture?

Can you imagine a plant tissue, in vitro culturing without a culture medium? Of course not! Culture medium either in the liquid, solid or semi-solid state is essential. Why? Because it provides support and the nutrients necessary for plant growth that are normally available in the soil. A culture medium is a complete mixture of organic and inorganic nutrients that, depending on your needs, can be supplemented with vitamins, amino acids, and plant hormones.

We have an interesting article explaining different ingredients present in a culture medium. You can read it here.

Currently, many formulations of culture media are available. The most widely used medium is the Murashige and Skoog (MS) invented in 1962. It is considered as the ‘standard medium’ and most of the crops thrive well in it.

There are also other media mixtures present in the market, such as Gamborg (B5) and Linsmaier and Skoog (LS) which are commonly used as well. You can read about them in detail here.

How can you prepare culture media for your experiment?

You can prepare it by dissolving the commercially available basal salts mix or by using self-prepared media ingredients according to a table of the component list. If you want to learn the basic steps of media preparation, this article is for you!

Getting started

When preparing your culture media, it is important to have a media preparation area arranged. In this area, you will prepare the culture media as well as store chemicals and glassware. The equipment we need for media preparation are:

• Hot plate and magnetic stirrer;
• Analytical and top-loading balances;
• pH meter;
• Refrigerator and freezer;
• Water purification and storage system;
• Equipment for sterilization of media and/or glassware (autoclave and/or media preparator);
• Glassware, spatulas, and chemicals.  

Before you begin preparing your media, make sure you have all of the materials you will need and that the preparation area is clean to reduce the possibility of contamination. It is generally recognized that various tissue culture errors are often caused by erroneous medium preparation. As a result, you must be cautious and precise.

Media preparation

Media preparation is a crucial step, and you need to develop a system for your step-by-step routine. Just imagine it as a cooking recipe!  

If you are starting in plant tissue culture, you can opt for commercially available powder mixes. Although each culture medium has a somewhat distinct preparation methodology, the steps outlined below detail how to properly prepare media for tissue culture:

1. Add 4/5 of distilled water of the total volume of media to be prepared in a beaker. For example, for 1 L of media, you add 800 mL of water.
2. Mix the powdered medium mixture with the water.
3. Add sucrose to the beaker. Here the quantity is based on the plant under consideration and also the media recipe you are following.
4. Adjust the pH using sodium hydroxide (NaOH) or hydrochloride acid (HCl). An ideal pH for plants is between 5.7 - 5.8. NaOH is used for increasing the pH value whereas HCl is used for decreasing the pH value of the solution.
5. If you are preparing a solid or semi-solid media, add agar or any other gelling agent to the beaker. The quantity of agar you need to use depends, among others, on the method of sterilization. For example, for MS medium to be sterilized in an autoclave would require 8 grams of agar while for an efficient media preparator, you would need 4-6 grams of agar.
6. Add growth regulators to the beaker which depends on the goal of your experimental plan.
7. Add the amount of distilled water required to make the total desired volume of media.
8. Sterilize the medium.
9. If you need to prepare a large volume of specific media recipes, then it is much more efficient to use a media preparator. In this case, all the ingredients are directly added to the media preparator, except for the growth regulators. You add these after checking and adjusting your pH level; usually around 60-70 degrees Celcius.

However, if you want to prepare your own media from scratch, you can follow these steps:

1. In a beaker, add ¾ of distilled water of the total volume of media you will prepare.
2. Add appropriate quantities of the stock solutions (each of macro-and micronutrients) according to the list of ingredients and check them off as they are added.
3. Add sucrose and vitamins.
4. Add the plant growth regulators and vitamins in the required quantities.
5. Adjust the pH of the medium using NaOH or HCl.
6. Add the gelling agent (If you want a solid or semi-solid medium).
7. Add the amount of distilled water necessary to complete the volume of the medium being prepared.
8. Sterilize the medium.

In both methods (using a powdered mix or stock solutions), the preparation of the media is done while stirring and using the hot plate to dissolve the components better (unless you are using a media preparator where stirring and heating are carried out inside the system automatically).

Both methods will deliver the same results if done well. Only with the exception that using powdered medium helps you save time, and labor and ensures all components are added in the right quantity. With stock solutions, you will have to be very precise with the weighing and concentration of the ingredients.

Stock solutions, why prepare them?

Stock solutions are concentrated solutions of chemicals that are prepared in advance and used to make several batches of media. The use of stock solutions reduces the number of repetitive steps in media preparation and hence, the chance of human or experimental error. To prepare them, components are often dissolved in distilled water to get the desired concentration. These solutions can be of different macro and micronutrients, vitamins, and growth regulators.  

Dispensing the medium

When the medium is ready, the next step is dispensing it into the vessels where the tissue cultures will grow. It is efficient to dispense after sterilization into pre-sterilized vessels inside a laminar flow. An alternative solution would be to autoclave the glass growth vessels with media.

The dispensing can be done in different ways, where, in our experience, the batch volume usually determines the level of automation. For small volumes, manual dispensing can be sufficient. However, when preparing larger volumes, for example with a media preparator, automatic dispensers can help you automate and optimize the dispensing process.

Ensuring success

The correct preparation of the culture medium will determine the success of your experiments. However, you can encounter difficulties such as precipitation and contamination. To avoid both, we give you the following tips:

• You should wear gloves, hair nets, mouth masks, and lab coats.  
• You should never pour excess stock solution back into the original stock solution container and never put excess sucrose or agar back into the original container.
• Do not forget to clean the spatula between weighing different chemicals.
• Make sure the glassware is clean and dry without soap or other chemical residues.
• All the media/stock solutions you prepare should be properly labelled indicating the type of stock/media and date of preparation.
• You should store media/stock solutions in the refrigerator and the dry powdered mix as well as other biochemicals at the appropriate room temperature, preferably in dark.
• You can use vessels with filters such as our inVenti⁺ which can significantly reduce the problem of condensation.

We hope this article serves you as a guide when preparing your tissue culture media!

For more interesting articles on different aspects of plant tissue culture, keep checking this space.

By Valeria Franco Franklin | 20 June 2022

References

• Chimdessa, E. (2020). Composition and Preparation of Plant Tissue Culture Medium. Tissue Cult Bio Bioeng, 3: 120. https://doi.org/10.29011/2688-6502.000020
• Saad, A. I. M. & Elshahed, A. M. (). Chapter 2 – Plant Tissue Culture Media. In Recent Advances in Plant in vitro Culture, 29-40. http://dx.doi.org/10.5772/50569
• Smith, R. H. (2013). Chapter 3 – Media Components and Preparation. In Plant Tissue Culture, 31–43. https://doi.org/10.1016/B978-0-12-415920-4.00003-7
• Phillips, G. C., & Garda, M. (2019). Plant tissue culture media and practices: an overview. In Vitro Cellular & Developmental Biology – Plant, 55: 242-257. https://doi.org/10.1007/s11627-019-09983-5
• Mohamad, N. I. & Wai, T. (2016). A Simple and Easy Method for Preparing Solid and Liquid Media for Plant Culture. In Experimental Methods in Modern Biotechnology, 9:15.