8 components of tissue culture media

Did you ever thought "where does plants in tissue culture get their nutrients from"? Let's answer this question for all of us: it's 'tissue culture media'! Tissue culture media is the source through which your plants receive nutrients for good and healthy growth. It is a combination of different components which provide different nutrients at different stages of plant growth.

It also plays an important role in deciding the success of tissue culture process for any plant species!

Tissue culture has been under practice since the last few decades. There has been discoveries of various fascinating possibilities of growing different plant species from a plant part or just fossils, simply by using tissue culture . Isn't this fascinating?

Plant tissue culture is currently under use not just for producing plants in large numbers for growing in different regions of the world. But it is also an important scientific practice to improve plant characteristics, for instance, developing a coffee variety with higher caffeine content.

However, plant tissue culture require extensive trial and error experiments. The success of an experiment does not only depend on using the right material and sterile conditions, but also on the composition of culture media. For every specific plant species and its varieties, we need to curate a different culture media recipe. These recipes also differ with different plant growth stages.

In other words, preparing a tissue culture media recipe is like cooking, however, with careful balance between different ingredients/components. Now, you might be wondering what components do you need to prepare tissue culture media? So in this article we are trying to answer this question for you!

There are 8 major broad components you need for preparing a tissue culture media recipe. Let us talk a bit about each component:

Macronutrients

Macronutrients are elements that plants need in large quantities (millimolar) for their growth and development. The major macronutrients used here are Nitrogen, Potassium, Phosphorus, Calcium, Magnesium and Sulphur.

Among these 'Nitrogen' plays the most vital role. It is essential for the growth of a plant in tissue culture as it’s important for amino acids, nucleic acid and proteins.

Micronutrients

Micronutrients are elements that are essential for good growth, however, plants need them in extremely small quantities. These include Boron, Manganese, Iron, Zinc, Copper, Molybdenum, Cobalt and Iodine.

Among these, 'Iron' is the most important microelement plants need. It is important for chlorophyll synthesis process, the source of green color we see in plants. It is also playing a role in conversion of energy during photosynthesis.

Each macronutrient and micronutrient has its own specific function in plant growth process. If you want to read more on these nutrients and their functions, then do check our article on "2 major nutrient components of culture media".

Carbohydrates

Plants also need organic chemicals such as sugars, starches and cellulose in tissue culture. Plants are unable to manufacture all the sugar they require to grow. The most common carbohydrate we need to use in tissue culture is 'Sucrose'.

Sucrose is also a common carbohydrate available in plants, but still require it additionally via culture media. There are other forms of sugar as well, such as sugar from sugarcane and beetroot, D-Mannitol and D-Sorbitol. We can use these other forms occasionally which depend on the requirement for certain plant species.

Vitamins

For healthy growth of tissue cultures, plants need vitamins in the media. Mostly, plants require the vitamins of B group as they act as coenzymes (molecules that help enzymes to perform their function).

Different vitamins used are thiamine (vitamin B₁), nicotinic acid (vitamin B₃), adenine (vitamin B₆), riboflavin (vitamin B₂), pantothenic acid (vitamin B₅), biotin (vitamin H), folic acid (vitamin M) and myo-inositol. The most important vitamins for your plants are 'Thiamine' and 'Myo-inositol'. All other vitamins are not essential and we need them in tissue culture for specific benefits only based on the requirements.

Amino acids

Plants need amino acids as the organic source of nitrogen which will be readily available for uptake. As amino acids combine and form proteins, we use them specifically for cell and protoplast culture. In a nutshell, cell and protoplast culture are the types of tissue culture where a plant is formed from a single cell.

Amino acids also help in shoot and root initiation. One of the simplest amino acid we know is 'Glycine' which we commonly need to preparing culture media. Other amino acids that we can use for different media recipes are L-glutamine, asparagine, serine and proline.

Plant growth regulators

Just like humans, plants also require hormones! Plants use phytohormones present in culture media to initiate different kinds of growth and differentiation of tissues to different organs and functions. Plants need these hormones in trace quantities. The hormone requirement also varies according to the plant variety, tissue type and tissue culture stage.  The plant growth regulators that plants frequently use are 'Auxins' and 'Cytokinin'.

What are the different phytohormones that plants require? Let us discuss briefly about them too!

Auxin

Auxins promote cell enlargement, adventitious bud formation and root initiation. They are also responsible for apical dominance and suppression of lateral bud formation as well.

The commonly used auxins are 'indole-3-acetic acid (IAA)', 'indole-3-butyric acid (IBA)', 'naphthalene acetic acid (NAA)' and '2,4-dichlorophenoxyacetic acid (2,4-D)'.

Cytokinin

Cytokinins promote cell division, axillary bud proliferation and short formation. Apart from these, it also helps in delaying the aging process (also called as 'senescence') and influence transport of auxins.

The commonly used cytokinins are 6-benzylaminopurine (BAP), 6-(y,y-Dimethylamino)purine (2iP), Kinetin, Zeatin and Thidiazuron (TDZ). Among these, the most effective cytokinin which we commonly observe in different media recipes is 'BAP'.

Gibberellin

Gibberellins promote elongation of internodes, meristem growth and developing in vitro embryos into normal plantlets. However, their use is not common for every media recipe in plant tissue culture. They can be detrimental to growth of your tissue cultures. So its always good to study if the plant under consideration requires gibberellins or not. 'GA₃' is the most commonly available gibberellin in tissue culture.

Ethylene and Abscisic acid

Some plants also require ethylene for their tissue culture, however it is rare. Ethylene is famous for its effect on fruit ripening, flowering and leaf abscission ('shedding of leaves'). Hence, it is not essential for your plants in early phases of growth and tissue culture.

Abscisic acid (ABA) plays a controlling part in stomatal closure (pores present on plant surface for gas exchange), bud dormancy and seed dormancy. This hormone can play both a role of stimulation as well as of inhibition of functions in plant growth. But this depends on the hormone concentration you use and also on the plant species you consider.

Antibiotics

In rare cases, you also need antibiotics in tissue culture media in order to deal with contamination and eradicate microorganisms. However, you should be extremely careful with their use! Antibiotics have the potential to kill your cultures, build up resistance or just induce genetic instability.

'Ampicillin', 'streptomycin', 'kanamycin' and 'cefotaxime' are some of the antibiotics that are common for use in tissue culture laoratories.

Gelling agents

You need gelling agents to transform the liquid media into semi-solid state. This ingredient is necessary as in liquid cultures, your explants would submerge and die due to anaerobic conditions (no available oxygen).

The commonly used gelling agents are 'agar', 'agarose', 'gellan gum' and 'isubgol'. However, for better growth of certain plants in micropropagation, for example Inca lily, media without gelling agents are preferred as they perform better in liquid media.

So these are the 8 components you need to prepare your tissue culture media. You can play with different combinations and concentrations for different components. You can experiment for different plant species in different combinations and see for yourself what works best!

We hope this article gave you more insight into the world of plant tissue culture. For more articles, keep checking this space!

By Nancy Bhatia | 04-May-2021

References

1. Kyte, Kleyn, et al (2013) Plants from test tubes: An introduction to micropropagation. Timber press, Inc.
2. Bhojwani, S.S., & Dantu, P.K. (2013). Plant Tissue Culture: An Introductory Text. Springer India
3. Robert H. Smith (2013). Plant Tissue Culture- Techniques and Experiments. Elsevier Inc.
4. https://www.intechopen.com/books/recent-advances-in-plant-in-vitro-culture/plant-tissue-culture-media
5. https://www.researchgate.net/publication/40103634_The_components_of_plant_tissue_culture_media_II_Organic_additions_osmotic_and_pH_effects_and_support_systems