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Though a number of techniques have been developed to culture isolated single plant cells, the following are those which are used frequently.

(a) Bergmann’s Cell Plating Technique:

This is the most popular technique of single cell culture. The density of the isolated single cells in suspension culture is balanced to twice the finally desired plating cell density. Melted agar (0.6-1%) containing medium of otherwise the same composition as the suspension culture medium is cooled and maintained at 35°C in a water bath.

Equal volumes of the two media are mixed and rapidly spread out in petri-dishes in such a manner that the cells are evenly distributed and fixed in a thin layer of the medium after it has cooled and solidified.

The petri-dishes are sealed with paraffin. Suspension cultures which carry cell aggregates in addition to isolated single cells should be filtered through a serve which allows only single cells or small cell aggregates to pass through. The large cell aggregates are discarded and only the fine suspension is plated.

The petri dishes are observed under the microscope in inverted position and the single cells are marked on the outside of the dish by a fine marker to ensure the isolation of pure single cell-clones. The culture containing petridishes are incubated in the dark at 25°C.

Suspension Culture:

When the pieces of calli are transferred to liquid medium and the medium is continuously agitated to obain isolated single cells, the culture is called ‘suspension culture’. Basically there are two types of suspension cultures, viz., batch culture and continuous culture.

(i) Batch culture when cultures are continuously propagated by routinely taking a small aliquot of the suspension and transferring it to a fresh medium, the suspension culture is called ‘Batch culture ‘.

(ii) Continuous culture when cultures are grown in culture vessels under steady state for prolonged periods by adding fresh medium and draining out the used medium, the suspension culture is called ‘continuous culture’.

(b) The Filter Paper Raft-Nurse Technique:

This technique has been developed by Muir et al (1954). It involves culturing isolated single cells on the top of an actively growing callus separated by a piece of filter paper. What is done actually is that the single cells are isolated from suspension cultures with the aid of a micropipette.

Several days earlier to this isolation, sterile 8 mm x 8 mm squares of filter paper are placed under aseptic conditions 6n the top of the established callus, called “nurse tissue”, of the same or different species.

The filter paper is wetted by liquid and nutrients from the nurse tissue. The isolated single cell is now placed on the wet filter paper raft. When a microscopic colony proliferates from the cell, it is transferred to suitable agar-medium for further growth and development of cell alone.

(c) The Microchamber Technique:

This technique has been developed by Jones et al in 1960. A drop of the nutrient medium carrying a single cell is taken out by micropipette from the suspension culture, is transferred on a sterile microscopic slide. A drop of sterile mineral oil is placed on either side of the medium-drop and a coverglass placed on each mineral oil drop.

A third cover glass in now taken and placed on the medium drop bridging the two coverglasses and forming a microchamber to enclose the single cell aseptically within the mineral oil. The latter does not allow any loss of water from the microchamber but allows exchange of gases. The whole slide in then placed in a petri-dish and incubated. When a microscopic colony develops from the cell, the cover glass is removed and the colony is transferred to fresh suitable medium for further growth and development of cell alone.