It was Haberlandt in 1890s that put forth the pioneering attempt to culture mechanically isolated mesophyll cells. He succeeded in maintaining these cells alive for about 10 days in culture but cells did not divide. He failed to achieve division of isolated cells. Since then, the progress in the field of isolating and culturing individual cells has been so spectacular that it has become possible not only to culture isolated single cells but also to induce divisions in such cells in complete isolation and, finally, to regenerate whole plants from them.
The most important aspect that deserves attention in isolated single cell culture technology is the initiation of division and proliferation of isolated single cells which starts only when they are placed into “conditioned medium” (medium in which the tissue has been grown for some time). Isolated single plant- cells resemble with animal cells in this respect.
However, a conditioned medium is prepared by culturing high densities of cells of the same or different species in fresh medium for a few days and then the cultured cells are removed by filter- sterlization making the medium free of them.
This medium now contains essential amino acids such as glutamine and serine as well as growth regulators such as cytokinins. When the isolated single cells are placed into the conditioned medium in exactly the same way as microorganisms, they initiate division and, instead of developing a colony as do the microorganisms, they proliferate to result in a callus.
The latter is induced to regenerate plants.
Isolation of Single Cells
(a) From Intact Plant Organs:
(i) Mechanical Isolation:
Leaf tissue is considered the most suitable material for single cell isolation. The widely used mechanical method for the isolation of mesophyll cells is the gentle grinding of leaves followed by cleaning the cells by filteration and centrifugation. Another device for the large scale mechanical isolation of leaf cells have been developed by Rossini (1969).
In this method the leaves are first surface sterlized by rapid immersion in 95% ethanol followed by rinsing for 15 minutes in filter-sterilized 7% solution of calcium hypochlorite. The leaves are now washed in sterile distilled water and cut into small pieces which are homogenized with culture medium (1.5 g of leaf material with 10 ml of culture medium).
The homogenate is filtered, the filterate is subjected to slow speed centrifugation which sediments the isolated cells. The supernatant is removed and the cells are transferred to suittable medium. Mechanical isolation of cells is considered advantageous at least in two aspects, over the enzymatic method. First, it eliminates the exposure of cells to the harmful effects of enzymes second, the cells need not be plasmolysed.
Composition of media recommended for the culture of isolated mesophyll cells (after Bhojwani and Rajdan, 1983)
(ii) Enzymatic Isolation:
Enzymatic isolation of plant cells is generally undertaken by plant physiologists and biochemists. In this method, the fully expanded leaves are taken from 60 to 80 day old plants. They are surface sterilized in 70% ethanol and rinsed in 3% sodium hypochlorite solution contain 0.05% Teepol or Cetavelon.
The leaves are now washed with sterile distilled water and their lower epidermis is peeled off with the help of fine forceps. A piece of peeled area is excised and transferred to enzyme solution. The enzyme, however, is infiltrated into the leaf-tissue with the aid of vacuum pump.
The material is incubated at 25°C on a reciprocating shaker with a stroke of 4-5 cm at the rate of 120 cycles/min. The enzyme solution is changed every half an hour. The solution collected after the first 30 minutes is discarded.
The solution collected after the second 30 minutes contains largely spongy parenchyma cells, and those after the third and fourth 30 minutes contains predominantly palisade cells. These isolated cells are washed twice with culture medium, and finally cultured.
(iii) Isolation from Cultured Tissue:
Traditionally, the single cell systems used in basic and applied researches are isolated from cultured tissue because this approach is convenient and widely applicable. Cultures are initiated by simply placing explants from surface-sterlized plant parts (e.g. leaves) on a nutrient medium containing phytohormones, viz. auxins and cytokinins.
The explants develops a callus which is separated and transferred to a liquid medium. The callus is agitated in the liquid medium. As a result, it breaks up in the medium and gives a suspension of isolated cells, small clusters of cells and much larger aggregates. When the isolated single cells are separated from the suspension culture and placed onto a suitable medium, they initiate dividing and proliferate to give a callus.