Protoplast Culture: Isolation. In the previous article, we have understood what is meant by plant tissue culture. In this article we will understand the meaning of protoplast and its isolation. Well, protoplast means a plant cell without its cell wall, but the question arises how will we take that cell wall away from a plant cell, well that's when the isolation part comes in, which is when two methods of protoplast isolation comes in. Let's understand.
What are Protoplasts
Protoplasts are naked/bare plant cells without the cell wall, but they possess plasma membrane and all other cellular components. Protoplast represents the functional plant cells but with the lack of the barrier, cell wall.
The cytoplasm, Nucleus, Rough Endoplasmic Reticulum, Smooth Endoplasmic Reticulum, Golgi apparatus, Mitochondria, Chloroplast, Vacuoles, Ribosomes, Cell Membrane, and the most awaited last one the “Cell Wall.”
So if we remove the cell wall the protective barrier of the plant cell all that we remain with is the cytoplasm of the cell, plant cell organelles, and Cell Membrane. So all we remain with is called Protoplast.
Protoplast= cytoplasm+plant cell organelles+cell membrane - cell wall
OR
Protoplast= Everything in the plant cell uptill the plasma membrane - cell wall
Historical Developments
The term protoplast was first introduced in l880 by Hanstein. The first isolation of protoplasts was achieved by Klercker (1892) using a mechanical method. A real beginning in protoplast research was in 1960 by Cocking who used an enzymatic method for the removal of cell walls. Rakabe and his associates (1971) was successfully able to achieve the regeneration of the whole tobacco plant from protoplasts. Rapid progress occurred after the 1980's in protoplast fusion to improve plant genetic material and the development of transgenic plants.
Protoplast
Protoplasts of different species can be fused together to generate a hybrid and this process is referred to as somatic hybridization (or protoplast fusion).
Cybridization is the phenomenon in which there is a fusion of a normal protoplast with an enucleated (without nucleus) protoplast that results in the formation of a cybrid or cytoplast (cytoplasmic hybrids).
The isolation, culturing, and fusion of protoplasts is a fascinating field in plant research. Protoplast isolation and their cultures provide millions of single cells for a variety of studies. Protoplasts have a wide range of applications as shown below.
Applications
l. A whole plant can be generated by culturing a protoplast.
2. From protoplast fusion Hybrids can be developed.
3. lt is easy to perform single cell cloning with the help of protoplasts.
4. Genetic transformations can be done through genetic engineering of protoplast DNA.
5. Protoplasts are brilliant materials for ultrastructural studies.
6. Isolation of cell organelles and chromosomes is easy from the protoplasts.
7. Protoplasts are useful for plasma membrane studies (transport and uptake processes).
8. Mutants isolation from protoplast cultures is easy.
How to remove the cell wall of a plant cell for obtaining Protoplast?
Well, it is very simple......
Isolation of Protoplast
Protoplasts are isolated by two methods as mentioned below:-
1 . Mechanical method
2. Enzymatic method
1 . Mechanical method= Protoplast isolation by the mechanical method is a tedious procedure. This method results in the isolation of a very small amount of protoplasts. The technique involves the following stages-
a) A piece of epidermis from a plant is selected.
b) The cells are subjected to plasmolysis. This leads the protoplasts to shrink away from the cell walls.
c) The tissue is dissected to release the protoplasts from the cell wall.
The mechanical method for protoplast isolation is no longer in use because of the following limitations as below-
a) The yield of protoplasts and their viability is low because while isolating protoplasts some cells might get damaged which leads in losing their viabiltity.
b) lt is restricted to certain tissues with vacuolated cell.
c) The method is laborious and hard to perform.
2. Enzymatic method= This method is very widely used technique for the isolation of protoplasts. The advantages of the enzymatic method include a good yield of viable cells and minimal to no damage to the protoplast. The enzymes that can digest the cell walls are required in this method. Chemically, the plant cell wall is mainly composed of cellulose, hemicellulose, and pectin which can be degraded by the enzymes cellulase, hemicellulase, and pectinase respectively. (cell wall is made up of cellulose, hemicellulose, and pectin so if we want to degrade the cell wall we must use these enzymes that will degrade these components )
This method can be performed in two ways as follows :-
A.
Two-step or sequential method
B. One
step or simultaneous method
1. Two-step or sequential method: The tissue is first treated with the pectinase (macerozyme) to separate cells by degrading middle lamella. These free cells are then treated with cellulase to release the protoplasts. Pectinase breaks the cell aggregates into individual cells while cellulase removes the cell wall properly.
2. One-step or simultaneous method: This is the most preferred method for protoplast isolation. This method involves the simultaneous use of both the enzymes - macerozyme and cellulose.
Now that we have Isolated the Protoplasts how should we check their Viability?
Viability of protoplasts
It is essential to ensure that the isolated protoplasts are viable so that they are capable of undergoing sustained cell divisions and regeneration. There are several methods to assess protoplast viability, as shown below.
a) Fluorescein diacetate (FDA) staining
method- The dye accumulates inside the viable protoplasts which can be detected by
fluorescence microscopy.
b) Phenosafranine stain is selectively
taken up by dead protoplasts (turn red) while the viable cells remain unstained, helping to distinguish between viable and nonviable cells.
c) Evans blue dye is excluded by intact membranes.
d) The ability of the protoplasts to undergo continuous mitotic divisions (this is a direct measure).
Culture of protoplast
After the isolation of protoplast and checking their viability, it's time for their culturing. Liquid culture is the preferred method for protoplast cultivation .B5 or Murashige and Skoog media (MS) is preferred.
Formation of cell wall
The process of cell wall formation in the cultured protoplasts starts within a few hours after isolation which can take two to several days. As the cell wall development occurs, the protoplasts lose their characteristic spherical shape of the cell. The recently developed cell wall by the protoplasts can be identified by using a calcofluor white fluorescent stain.
After development of the cell wall. Development of the callus/whole plant take place.....
Summary
CONCLUSION
Before we could directly dive into protoplast culture we understood in detail what is protoplast, and then we went through the isolation of protoplast in different ways. We checked the viability of protoplast cells and also cultured them. We discussed their applications as well
Reference:-
Biotechnology by U Satyanarayana.
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