Single cells are cultured to produce clones of plants, to study genetic modification as well to avoid the chimeric effect of tissue culture. Isolated protoplasts and single cells obtained from plant tissues are cultured using different techniques. The single-cell culture is difficult as plant cells require some metabolic products that are synthesized by cells present in close vicinity.
Single-cell culture uses
Single cells isolated from plant tissue are cultured to obtain clones of a single cell.
These single cells cultures are used for crop improvement programs,
for the generation of plant clones,
for the generation of genetically modified plants.
These are used in industry for the production of single-cell protein and other such uses.
Different techniques are used to successfully culture single cells.
There are different techniques are used to culture single cells
Cell plating technique
Micro chamber technique
Nurse callus culture technique
Micro droplet technique and
Paper raft nurse culture technique
Cell plating technique
Single-cell isolated from loose callus or explants plated in solid medium. For this technique, the single cells are adjusted for their concentration. The concentration can be from 1000 cells to 10000 cells per ml of medium.
Single cells are present in the suspension medium, this medium is diluted for the required concentration of 100 cells/ml.
15 ml of the single-cell suspension medium is added to an equal amount of agar medium which is cool down to 35 degrees Celsius to 45 degrees Celsius and single cells are added into this medium.
The medium is mixed gently to make the suspension into an agar medium. The medium is allowed to solidify.
This culture is incubated for 16 hours at 25 degrees Celsius under white light. Cells in the Agar medium start to divide and form small colonies of the cells.
The cells can form a small callus, the callus can be isolated and cultured into a rooting or shooting medium to obtain plantlets.
Micro chamber technique
A microchamber is prepared under aseptic conditions. For this technique, two drops of mineral oil are placed on the slide.
A microchamber is created on this slide between the two drops of mineral oil. In this microchamber is placed a suspension medium, that carries a single cell.
The two drops of oil are then covered with cover glasses these are called raisers, and a third glass is placed above these two glasses.
The oil prevents water loss but allows gas exchange. This microchamber is then placed inside the Petri plate and incubated.
A single cell present in the suspension medium divide and forms the callus of single cells is used to produce plantlets under suitable conditions.
Nurse culture technique
Under this technique, the actively dividing callus is irradiated to suspend cells in the dividing phase. This callus is producing phytohormone and metabolites that are required for the division and metabolism of single cells. The single cells are plated on this carpet of irradiated cells. The single-cell will divide and form colonies. Which will later develop into a Callus and this callus can be used for the development of plantlets.
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Micro drop technique
Under the microchamber technique, special microchambers are prepared on slides. For this silicon is used. Silicon creates small wells on the Petri plates. The wells are prepared of silicone, and a suspension medium is placed between the small wells, single cells are cultured in these prepared microwells.
Kao and Gleba and Hoffmann developed this method in the late 1970s. They used special Cuprak dishes that had two chambers of different sizes. The inner chamber had microwells. Single cells/ protoplasts are cultured in these wells. Outer chambers are filled with sterilized distilled water, to maintain humidity. It is covered by a disc and sealed with parafilm.
Micro drop method
This method was developed by Koop and Schweiger (1985). In this method, the protoplast or single cells are cultured in a drop of 1μl culture medium. this micro drop is developed by following steps.
1μl micro drops of 2M sucrose solution are dispensed on a cover glass.
This cover glass is overlaid with silica. Allow drying.
Rinse with water and sterilize under UV.
Dispense mineral oil 1microliter in these microchambers.
Dispense culture medium under the micro drop of mineral oil.
Keep the cover glass in a large chamber that is filled with sterile distill water. It will maintain humidity.
A whole plant can be obtained from single-cell culture, using this method.
Nurse raft culture technique
The single cells are cultured on a raft of filter paper. Actively dividing callus is separated from the single cells using filter paper.
The filter paper is dipped into a nutrient medium.
On the filter paper, single cells are placed.
The filter paper raft is placed on the callus.
The callus will provide metabolites required for the single cells to divide and develop into the colony.
The colony formed by single-cell on filter paper is used to form plantlets.
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