Protoplast culture: from single cells to whole plants

Plant and animal cells are different in many ways. One of the main differences is the presence of a cell wall in plant cells, something that animal cells lack. The cell wall is an outer protective layer that provides structural strength, support and regulates the transport of molecules within the cell. Thus, cell walls are key to all basic processes in plant physiology. However, did you know that you can obtain plant cells without cell wall?

Protoplast culture refers to the process in which whole plants are developed from the culture of cells without cell wall. This technique was discovered over 10 decades ago and is still widely used in plant breeding and crop improvement programs.

If you want to learn more about this technique and the benefits that it offers, this article is for you!

What is a protoplast?

In school, during your biology classes, you probably learned about plant cells and their parts. One of the most characteristic and important parts is the cell wall. It surrounds the cell and acts as a protective layer. So, if we think about the cell as a person, the cell wall would be like the clothes we wear. And, just as we can take off our clothes, the cell wall can also be removed from the cell.

When a living plant cell lacks a cell wall, it is called a protoplast. Basically, protoplasts are “naked cells” that have had their cell wall removed through mechanical means or by using degrading enzymes. They have a distinctive spherical shape and are surrounded by a plasma membrane that does not offer the same protection as the cell wall.

Protoplasts are of great interest because they are totipotent. It means that when cultured, they can grow, divide, and differentiate into a whole plant. They are sensitive, versatile and any type of plant, organ, and tissue can be used to produce them. Protoplasts are usually derived from leaf tissues, but callus, cell suspension, and pollen grains are also commonly used.

The first isolation of plant protoplasts was achieved in 1892 by Klercker in onion. Today, there is a protocol for protoplast production for nearly all crops.

Apart from protoplast culture, there are several other plant tissue culture techniques to develop healthy plants. You can read more about them in our article on "7 methods of plant tissue culture".

What is the need to produce protoplasts?

If the plant cell comes equipped with a cell wall, you may wonder, why produce cells without it? The cell wall is a really rigid layer that, when doing experiments, limits our insight of what happens inside the cell. So, to perform some experiments it is advantageous to work with protoplasts.

Due to the lack of a cell wall, protoplasts are a unique experimental system for studies of the structure and function of plant cells. Also, the removal of the cell wall allows plant protoplasts to take up micro- and macromolecules, viruses, bacteria, and genetic materials like DNA and nuclei. Due to this, they serve for:

1. Plant genetic transformation and cell modification studies;
2. Experiments studying plant virus infection; and
3. Introduction of genes of agricultural and horticultural interest to produce new varieties.

Plant protoplasts, after isolation, have the potential to reform the cell wall. So, they have been an important tool to investigate the mechanism of cell wall formation as well. Protoplasts can also be used for cell organelle isolation, cell cloning, and fusion.  The versatility of plant protoplasts has greatly facilitated the development of biotechnology, physiology, and modern botany.

The method of protoplast culture is useful for the large-scale regeneration of plants using single selected cells. Commonly, plants obtained through protoplast culture are non-chimeric, i.e. they come from a single cell type and show high somaclonal variation.

How to perform protoplast culture?

Protoplasts are, in fact, really useful biological systems for different applications. But how to produce them? There is no universal protocol for protoplast isolation and culture. Like everything in science, it depends on the plant species and the tissue from which the protoplasts are being extracted. General steps of a protoplast culture protocol are:

1. Stage 0 - Plant material: This involves the sterilization of the plant material, and the tissues are finely dissected or shredded. For a successful experiment, the use of young and fresh material is recommended.
2. Stage 1 - Protoplast isolation: It begins with the treatment of the plant material with a solution of degrading enzymes that remove the cell wall. Later, the protoplasts are removed from the solution, purified, and collected.
3. Stage 2 - Protoplast culture: It refers to the transfer of the isolated protoplast to an appropriate culture medium. There, these protoplasts develop the cell wall within hours, followed by cell division resulting in small cell colonies that will grow into callus. You can use liquid or semi-solid culture medium that needs to have a component such as mannitol that maintains a proper osmotic pressure.
4. Stage 3 - Plant regeneration: It can be achieved by organogenesis or embryogenesis. The former refers to the formation of tissues and organs while the latter refers to the formation of embryos. For this step, you need to move the callus to a medium that induces plant regeneration.

Among the different stages, different factors can determine the success of the protoplast culture, such as:

1. Plant source, genotype, age, and growth conditions;
2. Pre-treatment and enzyme concentration used;
3. Purification method; and
4. Culture medium (especially, plant growth regulators, osmotic stabilizers, and gelling agents) and growth conditions (temperature, light, and cell density).

Major limitations of protoplast culture

In practice, although protoplast culture is frequently used, you may encounter some challenges while performing it:

1. Protoplast culture is a process that demands specialized tissue culture expertise, requires complex manipulation, and can be time-consuming.
2. Protoplasts are very fragile and there is genetic instability associated with their culture.
3. Current methods for protoplast regeneration are very genotype-specific reducing its application and success.

However, the advantages of using protoplast culture instead of other techniques outweigh its limitations. Plant protoplasts have a wide range of applications from their use in the isolation of cell organelles such as chloroplasts and nuclei to developing new plant varieties as a result of the fusion between cells of different plant species. 

This fusion, known as somatic hybridization, is the most promising use of protoplasts. This advantage makes it possible to induce fusion between protoplasts of plants that do not normally hybridize sexually, producing somatic hybrid plants that exhibit novel nuclear and cytoplasmic genetic combinations!

We hope you got a glimpse of what is protoplast culture and why it is one of the promising methods in the plant tissue culture industry. For more informational posts on different methods of plant tissue culture, keep checking this space!

By Valeria Franco Franklin | 21-February-2022

About the author

Valeria Franco is from Colombia, the land of orchids. She is a focused and passionate biologist who specializes in biotechnology and molecular biology. Valeria has prior laboratory and research experience. She is presently employed as a content creator at Lab Associates and is always looking for new challenges. Valeria is enthusiastic about plant science themes and reading as a tool for lifelong learning. Her hobbies include studying foreign languages, traveling, and archery.

References

• Bhatia, S. (2015). Chapter 2 - Plant Tissue Culture. In Modern Applications of Plant Biotechnology in Pharmaceutical Sciences, 31-107. https://doi.org/10.1016/B978-0-12-802221-4.00002-9.
• Davey, M. R., Anthony, P., Power, J. B., & Lowe, K. C. (2005). Plant protoplasts: status and biotechnological perspectives. Biotechnology Advances, 23(2): 131–171. doi:10.1016/j.biotechadv.2004.09.008
• Pasternak, T., Lystvan, K., Betekhtin, A. & Hasterok, R. (2020).  From Single Cell to Plants: Mesophyll Protoplasts as a Versatile System for Investigating Plant Cell Reprogramming. Int J Mol Sci, 21(12): 4105. doi:10.3390/ijms21124195
• Purohit, S. D. (2012). Chapter 8 – Protoplast Culture. In Introduction to Plant Cell Tissue and Organ Culture, 161-170.
• Reed, K. M., & Bargmann, B. O. R. (2021). Protoplast Regeneration and Its Use in New Plant Breeding Technologies. Front Genome Ed, 3: 20. doi:10.3389/fgeed.2021.734951