Protoplasts Isolation

The protoplast, sometimes referred to as a naked plant cell refers to all the constituents in a cell of plant that are not the wall of the cell. The term”protoplast” was first used in the work of Hanstein in 1880 to refer to the living matter contained within the membrane of the plant cell. The protoplast that is isolated is unique because the plasma membrane’s outer layer is completely exposed and forms the only barrier between the outside surrounding environment and the inner living cells.

The separation of protoplasts out of plants was first accomplished using microsurgery of cell plasmolyzed by mechanical technique (Klercker 1892). However the yields were very poor and this technique is not very effective. The first enzymes were used was in the work of Cocking (1960) for the release of protoplasts. He isolated the cellulase enzyme from the flora of Myrothecium verrucaria that degrades cell wall.

Then, he used an extract of the cellulase enzyme to isolate protoplasts in the tomato root tips. Since then, numerous enzymes have been used to separate protoplasts. the most widely utilized enzymes are currently commercially available. Utilizing the cell wall degradation enzymes became recognized as the best method for separating large amounts of protoplasts that are uniform.

In the right conditions, in several species of plants the protoplasts are successfully cultivated to produce cell wall synthesis and, more recently, genetic modifications are also being developed. Protoplasts are beneficial for cell fusion research. They can also absorb foreign DNA cell organelles or viruses by their plasma membranes.

Isolation of Protoplasts

Protoplasts are isolated by two technique

  1. Mechanical method
  2. Enzymatic method

Mechanical Method of Protoplasts Isolation

In this way, huge and extremely vacuolated cells from tissues that store food like the onion bulb’s scales as well as the radish root, and beet root tissue can be used to isolate. Cells are then plasmolyzed within an iso-osmotic liquid that results in the ejection of the contents from the center of the cell. Then, the tissue is broken up and deplasmolyzed, releasing protoplasts that have been preformed.

Klercker (1892) removed protoplasts of Stratiotes aloides using this method. Mechanically, protoplasts were removed by gently pulling the new callus tissue arising from the expanded leaves of Saintpaulia Ionantha, which was grown in an auxin-rich environment to create thin cell wall. This method is not widely used due to certain drawbacks. It is restricted to specific tissues that have vast vacuolated cell populations.

The yield of protoplasts is typically extremely low. Protoplasts that are less vacuolated or high meristematic cells don’t exhibit the proper yield. The process is long and tedious. Because of the release of chemicals by damaged cells, viability of protoplasts is not high. Mechanical methods are beneficial when there are adverse negative effects of cell wall degradation enzymes.

Steps of Mechanical Method in Breif

  1. A small amount of epidermis of the plant is chosen.
  2. The cells are exposed to the process of plasmolysis. Protoplasts disappear from the cells’ walls.
  3. The tissue is cut to allow the protoplasts to be released.


  • Protoplasts have a low yield while their viability remains poor.
  • It is only restricted to certain tissues that contain vacuolated cells.
  • The procedure is time-consuming and tiring.

However, some people prefer mechanical methods when enzymes that degrade the cell wall (of an enzymatic process) can cause negative effects on protoplasts.

Enzymatic Method of Protoplasts Isolation

Enzymes were first used to separate protoplasts from plants in the green variety in the work of Cocking (1960). Cellulase was a component of the fungus Myrothecium verrucaria to extract protoplasts from the roots that belong to Lycopersicum the esculentum (Tomato). The mix of enzymes could be utilized to achieve optimal isolation (example an pectinase enzyme, a cellulase, and a hemocellulase). The plant enzymes employed can be classified into two types, which are:

  • Pectinases: break down the middle lamella and separate each cell.
  • Cellulase and hemicellulase: that decompose cells by breaking down the cells’ wall and letting the protoplast go.

The process of removing protoplasts using enzymes can be accomplished in two steps or as a single-step process.

  1. Two step or sequential method
  2. One step or simultaneous method

1. Two step or sequential method

It involves, as the name implies two distinct treatments that occur with two distinct stages. In the initial step macerase or macerozyme is a form of pectinase is used to breakdown the middle lamellae and let individual cells out of the tissue. The next step is when cell lines are cleaned using cellulase, which breaks down the cell wall and leaves protoplasts. The enzyme treatment has a shorter duration with the two-step procedure as compared to the one-step method.

2. One step or simultaneous method

This method is utilized more frequently than the two-step technique. This method is where tissues are mechanically loosen (e.g. through cutting the strips) followed by treatment using the mix of enzymes, namely, cellulase and pectinase. The two enzymes are mixed together for a longer amount of time.

Purification of Protoplasts

It is essential to cleanse protoplasts and get rid of isolating enzymes as well as the undigested tissues fragments and damaged protoplasts for productive production of protoplasts. In order to purify, the mixture of centrifugation, filtration and washing steps are utilized.

The protoplast-rich solution is then filtered through a metallic mesh or nylon (50-100 millimeters) to remove the larger portions of tissue that is not digested as well as cell clusters. Damaged cells and enzymes are eliminated through repeated centrifugation (3-10 minutes, 75-100 x kg) and resuspension with washing medium. If there are a large amount of residues present 20% sucrose may use to cover the washing medium. Protoplasts form a ring around the top layer of sucrose. Meanwhile, organelles and cell residues will remain in the lower part of the pellet in the test tubes for centrifugation.

Viability and density of protoplasts

It is crucial to test the viability as well as the amount of protoplasts isolated for experiments that follow the isolation. Ideally , the sample should have high viability and enough density of protoplasts that have been isolated. To test the viability of isolated protoplasts , several methods are employed:

  • The fluorescein diacetate (FDA) dyes viable protoplasts and can be seen fluorescing under a microscopes that are fluorescent.
  • Evans blue living protoplasts that do not allow the color pass through their membranes.
  • Red pigment that is neutral (concentrated only in cells that are active in metabolism)
  • Monitoring the flow of cytoplasm as an indication of an active metabolism
  • Calcofluor white or MR2 is the renewal of cells wall is observed using a fluorescent microscope.

Cultivation of Protoplasts

The most important factors for the success of the cultivation of protoplasts is the composition of the medium used for culture (especially the amount of sugars) as well as the temperature and the intensity of the light during incubation.

Liquid medium is generally preferred for the production of protoplasts due to its ability to control the osmoticum within the media, better control of cell density as well as a more simple change of culture medium , and the separation of cells during the cultivation process.

Protoplast Regeneration

The protoplasts isolated from the plant could be stimulated to regenerate, most preferably to produce a complete plant. There are however a some issues that can arise during protoplast regeneration. The protoplast can be damaged during the isolation process because of which their viability may decrease dramatically. Damage can be minimized by shortening the treatment of cells using degradative enzymes to isolate protoplasts. The conditions for culture should be designed for specific species. The conditions for regeneration must be customized for each species. This increases the amount of the protoplast that is regenerated. Each step should be meticulously planned and monitored to ensure the most efficient rate of regeneration , from the acquisition of the source through the cultivation conditions and the handling. Each chemical and reagent should be of high quality and the quantities should be designed to be optimal.

Factors Influencing the Isolation of Protoplasts

1. Plant material

Different organs and tissues of plants can be selected to produce protoplasts. The various organs that can be used to isolate of protoplasts are leaves stem apices, roots, shoots as well as hypocotyls, coleoptiles embryos, petioles and the pollen grains and calli suspensions of cells. The most reliable protoplast source is the leaf mesophyll. From these, a huge amount of cells that are uniform can be separated. In vitro plants may yield two times the amount of protoplast in comparison to in the vivo. The kind of material used as the source and the structure of its cell wall, the presence of an external layer, and the cementing substance between cells affect the isolation process in a significant way. The nature of the cell’s wall determines the enzymes used.

2. Enzymes

The composition and type of enzymes impact the procedure of isolation. The density and viability of isolated protoplasts are also contingent of the level, time, pH, and temperature of the treatment. The proportion of the enzyme solution to plant tissue can also affect the results of the isolation process.

3. Osmotic conditions

To prevent the osmotic shock that protoplasts may feel following the loss the cell wall within the solution surrounding it The osmotic pressure of the solution is adjusted to avoid this. The solution surrounding it could be the enzymatic mix either the washing solution, or the medium for culture. The solution is supplemented by sorbitol or mannitol as well as sucrose or glucose to regulate the Osmotic pressure. The protoplasts remain much more robust in a hypotonic state than in an isotonic solutions.

Importance of Protoplasts and Their Cultures

The isolation, growth and mixing of protoplasts is a fascinating area of plant research. Protoplast isolation and their culture are a source of millions of cells (comparable to Microbial cells) to be used in a range of research.

  • The protoplast that is in the culture can be transformed into a complete plant.
  • Hybrids can be created through protoplast-to-proplast the fusion of protoplasts.
  • It is very easy to do single cell cloning by protoplasts.
  • Genetic transformations are possible by the engineering of protoplast DNA through genetic engineering.
  • Protoplasts are a great material for studying ultra-structural structures.
  • The isolation of cell organelles and chromosomes is simple by separating protoplasts.
  • Protoplasts are helpful for membrane research (transport and uptake mechanisms).
  • The isolation of mutants from protoplasts is simple.


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