​Protoplast Culture: Isolation and Culture Methods (Part-1)

27th Apr 2022

​Protoplast Culture: Isolation and Culture Methods (Part-1)

Protoplast is defined as naked plant cells or plant cells without a cell wall. It consists of plasmalemma containing all the other cellular content or components in it. In tissue culture labs it’s used to regenerate a whole plant providing suitable artificial medium and environmental conditions. This process is known as protoplast culture.


Protoplast is defined as naked plant cells or plant cells without a cell wall. It consists of plasmalemma containing all the other cellular content or components in it. In tissue culture labs it’s used to regenerate a whole plant providing suitable artificial medium and environmental conditions. This process is known as protoplast culture.

The protoplast term was first introduced by the scientist Hanstein (1880). And, its first isolation was done by Klercker (1892) using a mechanical method. However, the serious efforts in the field of protoplast culture started in 1960, when a scientist named Cocking isolated the protoplast using enzymatic techniques.

Protoplasts of different species are generally fused to produce hybrid plants. The process is known as somatic hybridization (or protoplast fusion). Often a normal protoplast of the plant is also fused with a protoplast without a nucleus (enucleated protoplast) to form a cybrid or cytoplasmic hybrid. The process is known as cybridization.

In tissue culture, protoplasts are first isolated using mechanical and enzymatic methods and then cultured on an artificial liquid or semisolid agar medium.

This article presents a bring on the isolation techniques of protoplasts and how they are cultured in labs for the regeneration of plants.

Protoplast Isolation and Culture

Protoplast culture involves the following culture events:

  • Leaves of the specific plant are taken and sterilized.
  • The epidermis layer of the leaf is peeled and the leaf is cut into small segments.
  • The leaf segments are put into an enzyme mixture for protoplasts' release.
  • Then, the enzyme solution is collected and suspended in a washing tube, which is centrifuged.
  • The obtained pellets are resuspended in a sucrose washing medium and centrifuged again.
  • The protoplasts are separated at the top of the tube in the form of a band.
  • The protoplast bands are suspended in a culture medium.
  • The protoplasts are isolated and indued for wall formation.
  • After the wall formation, the cells enter into the division phase, forming a clump of a few tissues followed by callus formation.
  • Shoots are differentiated in callus and then plantlets are regenerated leading to the forming of a whole plant.

Figure: A detailed schematic diagram of the process of protoplast culture.

Protoplast Isolation

Protoplasts can be isolated from different parts of the plants including, roost leaves, stems, microspores, embryos, and fruits. However, among all these sources, the mesophyll tissues of the expanded leaves are the most preferred source of the protoplast isolation.

Protoplasts in labs are isolated mainly using two techniques:

  • Mechanical Method

In this method, a small piece of the epidermis of the plant is taken and subjected to plasmolysis, causing protoplasts to shrink away from the cell wall. Then the tissue is dissected for the release of the protoplasts.

This method of protoplast isolation is a tedious process and only helps in the isolation of only a few protoplasts. The other limitations are that the viability of the yielded protoplasts is low. However, still, the technique is preferred by some labs because of the deleterious effects of the enzymes used in the enzymatic approach to protoplast isolation.

  • Enzymatic Method

It’s the most widely used technique of protoplast isolation. In this method, protoplasts are isolated from the source using enzymatic solutions. This technique is faster, efficient, and releases more number of viable protoplasts without any damage.

The plant cell wall is composed of cellulose, hemicellulose, and pectin, thus, the enzymes used for protoplast isolation include cellulase, hemicellulase, and pectinase. The incubation period of protoplast sources in enzymes depends on the type of enzymes used to prepare the solution.

There are two ways of isolating protoplasts using the enzymatic technique. It includes:

  • Sequential Method: It involves the use of two enzymes, pectinase, and cellulase, First, pectinase separates cells from middle lamella, and then cellulase separates the protoplast from the rest of the cell wall.
  • Simultaneous Method: In this method, both macerozymes and cellulase are used at the same time for complete protoplast isolation.

Protoplast Purification

The separated protoplasts, using mechanical or enzymatic techniques are put under the purification process. Here, all the undigested cells, undigested tissues, and damaged protoplasts are filtered out.

After filtration, the obtained protoplasts are centrifuged, washed, and recovered above Percoll.

Viability of Protoplasts

For successful culturing and increased yield, it’s essential to test the viability of the protoplasts. Some of the methods are mentioned below:

  • Fluorescein diacetate (FDA) staining method
  • Phenosafranine staining method
  • Calcofluor white (CFW) staining method
  • Oxygen Uptake measurement technique
  • Method Involving the Measurement of Protoplasts’ Photosynthetic Activity

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Happy Culturing!!

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