In this article we will discuss about:- 1. Sources of Explant for Protoplast Isolation 2. Methods of Protoplast Isolation 3. Purification of Protoplasts 4. Isolation of Sub-Protoplasts 5. Protoplast Viability Test.
Each plant cell has a definite cellulose wall which is attached with each other by middle lamella made up of pectin. Protoplast is the content which is surrounded only by plasma-lemma. So to get the isolated protoplast it is essential to remove the pectin material to obtain the single cells and then to remove cell wall by enzymatic method.
Sources of Explant for Protoplast Isolation:
Protoplasts can be isolated directly from the different parts of whole plant which bears the soft parenchymatous tissue (e.g., young fully expanded soft leaves) or indirectly from the in vitro grown plant tissue (e.g., callus tissue). Protoplast yield and viability depends greatly on the type of tissue material from which it is isolated and the method used. Cell suspension cultures may also provide a very good source of protoplast.
Before isolation of protoplast, the source material if it is from in vivo grown plant then it should be properly surface sterilized using the proper method of sterilization. Then any of the methods either mechanical or enzymatic can be used to isolate the protoplast.
Methods of Protoplast Isolation:
The essential step of protoplast isolation is the proper use of osmoticum. The plant protoplast is an osmotic system where the plasma membrane acts as the semipermeable membrane which equilibrates the outward and inward flow of water. If the inward pressure is more than due to heavy inflow of water the protoplast will burst.
So during protoplast isolation the enzymes applied to isolate the protoplast and the tissue material should be placed in proper osmolyticum or plasmolyticum. Generally mannitol, a sugar alcohol at high concentration, an easily transportable component provides a good stable osmotic environment which prevents bursting of protoplast (Fig. 20.1).
Protoplasts may be isolated by any one of the two following ways:
(a) Mechanical method (Non-enzymatic method);
(b) Enzymatic method.
(a) Mechanical Method:
Any soft parenchymatous tissue is kept in a plasmolyticum. The plasmolysed tissue is then finely chopped into pieces and the intact cells (plasmolysed) are released into the medium from the cut surface. The suspension is then allowed for deplasmolysis and the released protoplasts attain their original size. The yield of protoplast in this method is very low, for large scale of protoplast yield the enzymatic method is followed.
(b) Enzymatic Method:
Young fully expanded soft leaves, or in vitro grown callus tissue or cell suspension culture grown cells can be used as the source material. The tissues or cells are incubated in plasmolyticum for 1 hr before enzymatic treatment. The intact tissue materials cut into smaller pieces to increase the surface area of enzymatic activity. The enzymes can be used either sequentially in two step method or in a single step by mixed enzymatic method.
The enzymes used are of three main categories:
The concentration of enzymes used and the time period of incubation varies greatly depending on the tissue type. In two step method, the pectinase and hemicellulases are applied first and then cellulase is applied for complete removal of cell wall.
After release of protoplast into the suspension, for removal of enzymes the protoplasts are collected in centrifuge tube as pellet and washed several times with the osmoticum.
Purification of Protoplasts:
For purification, the protoplasts suspended in osmoticum are centrifuged using sucrose (20%) solution. The viable protoplasts float on the top surface of sucrose solution forming a band. These protoplasts are then collected, re-suspended in osmoticum and washed several times.
Finally the protoplasts suspended in a measured volume of protoplast culture medium after counting the number with the help of hemocytometer. The viability of protoplast is checked with the help of Fluorescin diacetate staining or phenosafranine or calcofluore white.
Isolation of Sub-Protoplasts:
Sub-protoplasts do not contain the entire content of plant cells, these can be of different types such as cytoplasts, karyoplasts or mini-protoplasts or micro-protoplasts or microplasts.
(i) Cytoplasts lack the nucleus and contain the entire cytoplasm of a cell or part of it. These are often used for cybrid production.
(ii) Karyoplasts or Mini-protoplasts contain a nucleus surrounded by some cytoplasm and the original outer plasma membrane.
(iii) Micro-protoplasts contain fraction of both nuclear and cytoplasmic material.
(iv) Microplasts lack the nuclear material and contain only fraction of cytoplasm and outer membrane.
Protoplast Viability Test:
The most frequently used staining methods for checking protoplast viability are:
1. Fluorescein diacetate (FDA) dissolved in acetone is used at a conc. of 0.01% and intact viable protoplasts only fluoresce when observed under UV.
2. Phenosafranine is also used at a conc. of 0.01 %, which is specific for dead protoplast that shows red in colour.