Summary

Agar is one of the most widely used gelling agents in plant tissue culture, providing a semi-solid environment that supports explants while keeping essential nutrients and water available. In this guide, we explore what agar is, how it is produced, how it compares with other gelling agents like gellan gum, how much to use, how to prepare agar-based culture media, and how to troubleshoot common agar-related problems.

Introduction

Solidifying agents are one of the crucial components of tissue culture media. They offer solid support for the proper growth and development of plants when added to the media. Further, the use of solidifying agents also aids in the uniform distribution of nutrients, hormones, and other essential elements in the culture medium.

The two most common solidifying agents used in the process are agar and gellan gum. Agar is a polysaccharide derived from seaweed. It provides a gel-like consistency to the medium, allowing plant cells to adhere, grow, and differentiate.

Gellan gum is another solidifying agent used in plant tissue culture. Similar to agar, gellan gum is a polysaccharide produced by the bacterium Sphingomonas elodea. It is often utilized as an alternative to agar in plant tissue culture media.

Both agar and gellan gum can be used to solidify tissue culture media, but they differ in gel strength, clarity, concentration requirements, and other properties.

In this article, we'll explore what agar is, how it is produced, how different types and grades of agar differ, how agar is used in plant tissue culture, and what to consider when selecting and preparing agar-based media.

Basics of Agar

Agar is a solidifying agent and a jelly-like substance used in the preparation of tissue culture media. Chemically, it’s a gelatinous substance that is extracted from seaweeds. Agar is composed of two components: agarose and agaropectin. Agarose is a linear polymer of repeating units of agarobiose.

Agarobiose is a disaccharide that consists of D-galactose and 3, 6-anhydro-L-galactopyranose.

Agar is a very useful substance that has applications from food to laboratories. There are many types of agar, and all have different applications.

Why Is Agar Used in Plant Tissue Culture?

Agar plays several important roles in plant tissue culture:

• Physical support: Agar creates a semi-solid matrix that holds explants in place and provides a stable surface for shoots, roots, and other tissues to develop.
• Water and nutrient availability: The gel retains water while keeping dissolved nutrients, sugars, and other components of the culture medium available to plant tissues.
• Adjustable firmness: The concentration and type of agar can influence the firmness of the culture medium, allowing culturists to adjust the physical environment for different applications.
• Consistency: Tissue-culture-grade agar is designed to provide more consistent gelling performance, which can be important when reproducibility matters.
• Convenience: Agar is relatively easy to incorporate into culture media and has been widely used in plant tissue culture for decades.

How Does Agar Form a Gel?

Agar has a useful property that makes it particularly suitable for tissue culture: it can be dissolved by heating and forms a gel as it cools.

The gelling behavior comes primarily from agarose, one of the major components of agar. When agar is heated in water, its polymer chains separate. As the solution cools, these chains reassociate and form a three-dimensional network that traps water within the gel.

The concentration and characteristics of the agar influence the final firmness of the medium. This is why two agar products used at the same concentration may not necessarily produce identical gels.

For plant tissue culture, this matters because the medium needs to be firm enough to support the explant without creating an unnecessarily hard environment.

A Brief History of Agar

Agar was discovered by Tarazaemon Minoya of Japan in 1658. It was the first gelatin product to be introduced as food in the Far East over 300 years ago. Because of its utilization as a foodstuff, it was introduced in several other oriental countries.

Agar is extracted from the red algae (Rhodophyceae) members Gelidium and Gracilaria. In 1859, for the first time, the application of agar was recognized by Western people as a Chinese foodstuff.

In 1882, Robert Koch identified it as an important asset to grow bacteria in the lab. Thus, the manufacturing of bacteriological agar was initiated.

 

Manufacturing of Agar

Agar was initially extracted from Gelidium amansii, but extensive use created a supply crisis. Now, scientists had to search for other sources of agar. This is when Gracilaria seaweed started to be used. However, it was producing a very poor quality agar which couldn’t gel properly. This agar was named agroids.

Then Yaganwa introduced the solution for this problem. He transformed the agar obtained from Gracilaria into a strong final product using the alkaline hydrolysis method.

Now, the two techniques available for agar manufacturing include the freezing-thawing method and the Syneresis method. In the freezing-thawing method, the extracted agar from algae is frozen in a freezing tank.

Then, they are thawed and strained, which makes the agar more concentrated than before. The syneresis method is an advanced and modern technique of manufacturing agar. In this technique, the extracted agar is packed in a cloth with a closed mesh. Then, a horizontal hydraulic pressure is applied to remove water and other impurities from the agar. 

The syneresis method is more cost-effective and utilizes less energy than the freezing-thawing method. Agar produced by the syneresis technique has higher purity than that produced by the freezing-thawing method.

The dry weight extract obtained after pressing is generally 20% in the syneresis method and 11% in the freezing-thawing method.

Given below is a list of typical gel temperatures extracted from different agarophytes.

S. No.	Algae	Temperature
1	Gelidiella	42-45℃
2	Gracilaria	40-42℃
3	Gelidium	36-38℃
4	Pterocladia	33-35℃

Agar derived from different seaweeds can have different gel characteristics. These differences can affect how the agar behaves during preparation and use. However, the properties of a final agar product depend not only on the source seaweed but also on processing and purification.

Why Does Agar Processing Matter?

The way agar is processed can influence the characteristics of the final product. Factors such as the source of the seaweed, extraction process, purification, and processing conditions can affect properties including gel strength, clarity, purity, and consistency.

For plant tissue culture, these properties matter because a culturist needs a gelling agent that performs consistently from one preparation to another. This is one reason agar products designed specifically for tissue culture can be preferable to products intended for completely different applications.

Types and Grades of Agar

Not all agar products are the same. Different grades are manufactured and characterized for different applications, and their properties can vary depending on factors such as purity, gel strength, clarity, and processing.

Some common categories include:

Food-grade agar

Food-grade agar is primarily manufactured for use as a gelling, thickening, or stabilizing ingredient in food products. Although it can form a gel, its specifications are designed around food applications rather than plant tissue culture.

Microbiological-grade agar

Agar used in microbiology is formulated and selected for preparing culture media for microorganisms. Different microbiological media may contain agar as their solidifying component along with nutrients and other ingredients required for specific organisms.

Tissue-culture-grade agar

Tissue-culture-grade agar is intended for plant tissue culture and other biological applications where consistent gelling performance is important. Product characteristics such as gel strength, purity, and consistency can influence how the medium performs during culture.

Agarose

Agarose is the purified gelling fraction of agar. It is commonly used in molecular biology applications, including electrophoresis, and should not be treated as interchangeable with standard tissue-culture agar.

For plant tissue culture, choosing a product intended for tissue culture can help reduce variability in medium preparation and gel performance.

How Much Agar Should You Use for Plant Tissue Culture?

There isn't one universal agar concentration that works for every tissue culture system. The appropriate amount depends on the agar product, its gelling strength, the culture medium, and the plant material being cultured.

Agar concentration is usually expressed as grams per liter (g/L) of culture medium. Increasing the concentration generally produces a firmer gel, while reducing the concentration produces a softer gel.

For example, PCT's Supreme TC Grade Agar is recommended at 6–8 g/L according to its product instructions. Always check the recommended concentration for the specific agar you're using rather than assuming that the same concentration will produce the same gel with every product.

When optimizing an agar concentration, consider:

• The gelling strength of the product

• The desired firmness of the medium

• The plant species and explant type

• The stage of culture

• The composition of the basal medium

More agar isn't necessarily better. The goal is to achieve a medium that provides adequate support without making the gel unnecessarily firm.

Common Agar Problems in Plant Tissue Culture

Even when the basic preparation steps seem straightforward, agar-based media can sometimes behave differently than expected.

Why isn't my agar solidifying?

A soft or liquid medium can have several possible causes, including:

• Too little agar was added.
• The agar was not completely dissolved.
• The formulation or other medium components are affecting gel formation.
• The agar product has different gelling properties than expected.
• Preparation or sterilization conditions were not appropriate for the product.

If the problem occurs repeatedly, check the agar concentration, product specifications, preparation process, and storage conditions before changing several variables at once.

Why is my agar too hard?

A medium that is excessively firm may contain too much agar for the particular application. Agar concentration can be adjusted to achieve the desired gel firmness, but the appropriate range depends on the specific product and culture system.

Why does my agar look cloudy?

Agar does not always produce a completely transparent gel. Appearance can depend on the type and grade of agar, its purity, the medium formulation, and preparation conditions.

If high optical clarity is particularly important for your application, gellan gum may be worth considering as an alternative gelling agent.

Why does my agar vary between batches?

Differences in agar product, concentration, preparation, medium composition, or sterilization conditions can all contribute to differences in gel characteristics.

For reproducible tissue culture, using a consistent product and preparation protocol can help minimize variability.

Applications of Agar

Agar is used across several scientific, food, and industrial applications.

• Plant tissue culture: Agar is widely used as a gelling agent in plant tissue culture media, providing physical support for explants and developing plant tissues.
• Microbiology: Agar is used to solidify microbiological culture media, allowing microorganisms to grow as colonies on a stable surface.
• Food applications: Because of its gelling and water-binding properties, agar is widely used as a food ingredient in products such as jellies, desserts, and other formulated foods.
• Insect and animal research: Agar can also be incorporated into artificial diets and culture systems used in laboratory research involving insects and other organisms.

Its versatility comes from its ability to form stable gels while retaining water and dissolved components.

Agar VS Gellan Gum: What's the Difference?

Agar isn't the only option for solidifying plant tissue culture media. Gellan gum is another commonly used gelling agent and is often chosen as an alternative to agar.

Neither is universally better than the other. The appropriate gelling agent depends on the plant species, culture stage, medium formulation, desired gel properties, and the needs of the culturist.

Agar is often chosen for its familiarity, availability, and versatility, while gellan gum may be preferred when a clearer or particularly firm gel is desired.

Looking for Tissue-Culture-Grade Agar?

Choosing the right gelling agent can make a difference when you're trying to maintain consistent tissue culture conditions.

PCT's Supreme TC Grade Agar is designed specifically for plant tissue culture applications and can be used to solidify a range of culture media.

If you're troubleshooting inconsistent gel strength or looking for a reliable agar for your tissue culture protocols, explore the product specifications and recommended usage before making your selection.

[Explore Supreme TC Grade Agar →]

PCT also provides a range of tissue culture products, including MS media, gellan gum, PPM™, culture vessels, and laboratory equipment.

So, visit plantcelltechnology.com today and learn more about our products and services and how they help you excel in your tissue culture processes.

Happy Culturing!

Frequently Asked Questions

What is agar used for in plant tissue culture?

Agar is primarily used as a gelling agent to turn liquid culture media into a semi-solid environment that provides physical support for plant explants and developing tissues.

How much agar do I need for tissue culture?

The required concentration depends on the agar product and culture system. PCT's Supreme TC Grade Agar, for example, is recommended at 6–8 g/L according to its product instructions.

Why isn't my tissue culture agar solidifying?

Possible causes include insufficient agar, incomplete dissolution, differences in agar grade or gelling strength, medium composition, or preparation conditions.

Is gellan gum better than agar?

Not necessarily. Both can be used as gelling agents, but they have different properties. The best choice depends on your culture system and the characteristics you need from the medium.

Can I use food-grade agar for tissue culture?

Food-grade agar may form a gel, but it is manufactured for food applications and may have different specifications from tissue-culture-grade agar. For reproducible tissue culture, a product intended for tissue culture is generally a more appropriate choice.

What is the difference between agar and agarose?

Agar is a mixture containing agarose and agaropectin, while agarose is the more purified gelling fraction of agar. Agarose is commonly used in molecular biology, while agar is widely used for solidifying plant tissue culture media.

References

1. Armisén, R., Gaiatas, F. (2009). Agar. Handbook of Hydrocolloids, 82–107. doi:10.1533/9781845695873.82
2. https://www.sciencebuddies.org/science-fair-projects/references/grow-microbes-agar
3. https://www.britannica.com/topic/agar-seaweed-product
4. https://study.com/academy/lesson/what-is-agar.html