Protoplast isolation
Plant cells have a cell wall around their protoplast. The cell wall is composed of cellulose and pectin. This makes the fusion of plant cells difficult in vitro. It is also difficult to transfer genes to intact plant cells. So, to overcome this barrier plant cell is isolated from the cell wall. The isolation of protoplast is done through the mechanical method or enzymatic method.
Mechanical method
The mechanical method involves cutting the plasmolyzed cell. Thus releasing the viable protoplast. This method is highly inefficient as the yield of the protoplast is very low. Only vacuolated cells can be isolated through a mechanical method. Klecker used this method in 1892.
Enzymatic method
In 1960 Cocking demonstrated enzymatic isolation of protoplast. He used cellulase enzyme to isolate the protoplast.
The successful isolation of protoplast requires cellulase and macerozyme. The commercial preparation for protoplast isolation was made by Takebe et al in 1968.
Sequential method( Takebe et al)
The enzymatic isolation of protoplast can be done through the sequential method and through the simultaneous method. In the sequential method cellulase and Macerozyme are used sequentially. The macerozyme dissolves pectin of the middle lamina, which frees the plant cells from each other. The cellulase dissolves the cellulose cell wall to free the protoplasts.
Simultaneous method( Power and Cocking)
However, protoplast can be separated using both of these enzymes together. This method is called the simultaneous method of protoplast isolation. The protoplast can be isolated from nonlignified cells mesophyll cells. Sometimes cellulase, pectinase, and hemicellulase are also used for enzymatic isolation of protoplast.
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| Explants, Enzyme solutions A and B, isolated protoplasts |
Callus and non-embryonic suspension culture, female gametes can be used for isolation of protoplast.
Factors affecting protoplast isolation
Different factors affect the isolation of protoplasts. The viability is one of the most important concerns of protoplast isolation. So, some modification to enzymatic treatment has been used. These modifications include pretreatment for protoplast isolation, choice of source, and use of osmoticum for keeping the protoplast viable during enzymatic isolation. The isolated protoplasts are studied for their viability, regeneration of cell walls. These are also important factors in protoplast isolation and culture. However, the protoplast culture is similar to the single-cell culture.
Choice of source material
The Source of material for protoplast isolation can be mesophyll cells, as they are loosely arranged. They can be isolated from active callus. Protoplasts are difficult to be isolated from seeds of cereals. From cultured material, protoplasts are isolated from the cells that are in the log phase of division.
Pre-treatment for protoplast isolation
Mechanical treatment is done of tissue for protoplast isolation when protoplasts are isolated from mesophyll cells of the leaf. The source material is treated under aseptic conditions. It is sterilized and then washed with water. It is peeled to remove the epidermal tissue. Gentle brushing and cutting the leaf into small pieces also increase yield, as it increases surface area for enzyme action.
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| Peeled and cut explant is incubated in an enzyme solution and osmoticum ( 0.5%macerozyme, 2% cellulase, 13% sorbitol/ mannitol at pH 5.4 |
Enzymes for protoplast isolation
Common enzyme treatments are cellulase, pectinase, and hemicellulase. The commercial preparation of enzymes is available, such as Onizuka cellulase SS, Onizuka macerozyme SS. Pectolyase Y 23 is a highly powerful macerozyme in combination with cellulose, it releases protoplast from mesophyll cells of pea (Nagata and Ishii) 1979). Sometimes enzymatic treatment required cellulase, pectinase as well as hemicellulase.
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| Protoplast isolation steps- 1 explant sterilization, 2 peelings, 3,4 cutting and |
pH and temperature for enzymatic isolation
pH for enzyme activation is adjusted as 4.7-6 and the temperature for good enzyme action is 40 degrees Celsius to 50 degrees Celsius but for protoplast viability that temperature is adjusted as 25 degrees Celsius to 30 degrees Celsius. The incubation period is 30 minutes for an enzyme treatment.
Osmoticum
The isolated protoplasts are very fragile, to protect them osmoticum stabilizers are used in enzymes solution. This solution is slightly hypertonic. Sorbitol and mannitol are used as osmoticum for enzymatic isolation of protoplasts. The CaCl2 also improves protoplast isolation.
Ionic osmoticum is also used for enzymatic isolation of protoplast. Here KCl and MgSO4 are used as osmoticum.
Purification of protoplasts
Once protoplasts are isolated with an enzyme the next step is to purify protoplast from the osmoticum. For this, the large debris is squeezed and removed by hand. Then the remaining solution is filtered through a nylon sieve or metal sieve. The protoplasts are then present in the solution and these are then isolated through centrifugation. The solution is centrifuged at 100 x g for 10 minutes. This will result in the formation of the palate, the palate has protoplasts.
The palate is dissolved in the washing solution and centrifuged at 50 x g for 5 minutes. Protoplasts are then isolated through differential centrifugation. These are loaded on a sucrose pad( 21%) above the enzymatic solution. They form a clear layer at the junction of the solutions. Protoplasts are then taken by pipette and then washed again 2-3 times to obtain pure protoplasts. Sucrose and sorbitol are also used to form gradients for protoplast purification.
Viability test
Freshly isolated protoplasts are tested for their viability. this can be done by many methods. The viability of isolated protoplast can be established by observing cytoplasmic streaming of protoplast under the microscope.
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| Isolated protoplasts |
Measuring oxygen uptake by the protoplasts using oxygen electrodes.
Through exclusion staining by Evans blue dye is done to exclude dead protoplasts. Fluorescein diacetate is used to stain viable protoplasts.
Protoplast culture(1)
Viable protoplasts are cultured like the single-cell culture they can be cultured into a suspension medium to obtain callus or they can be plated in a nutrient agar medium through Bergman's technique of cell plating. Protoplast can also be cultured on agar beads. These techniques are given in single-cell culture methods in detail.
Significance of protoplast isolation
Isolated protoplasts are used to form cybrids and somatic hybrids.
These are used for physical gene transfer experiments.
Protoplast fusion can be done for sexually incompatible organisms.
Protoplasts are used to study the effect of drugs.
These are used for physiological studies.
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