In this article we will discuss about single cell culture in plants, explained with the help of suitable diagrams.
The single cells can be isolated from a variety of tissue and organ of intact plant or from callus tissue or from cell suspension. Single cells can be isolated from intact plant tissue or callus tissue by using either mechanical method or chemical method.
Mechanical isolation means chopping of the tissue material by fine scalpel which releases few intact single cells or by glass homogenizer the tissue may be crushed where the homogenate containing only few intact cells can be cultured. In chemical method the macerozyme or pectinase can be used to dissolve the middle lamella and releasing single cells from intact tissue.
In most cases the single cells are isolated from friable callus tissue or cell suspension culture. The cell aggregates or clumps are removed from the suspension by using mesh only and then the isolated single cells in the filtrate can be cultured either in liquid or in solid media.
Methods of Single Cell Culture:
There are various methods of single cell culture (Fig. 17.4A-C):
(a) Paper raft nurse culture technique
(b) Micro-chamber technique
(c) Micro-droplet technique
(d) Nurse culture technique
(e) Cell plating technique.
(a) Paper Raft Nurse Culture Technique:
The isolated single cells are placed aseptically on nutrient medium soaked filter paper and placed on a actively growing callus tissue. After small aggregate formation those are transferred to fresh media.
(b) Micro-Chamber Technique:
With the help of paraffin oil and cover glass a micro-chamber is formed on a glass slide and droplet containing single cells in medium is placed inside this micro-chamber and incubated for division.
(c) Micro-Droplet Technique:
In this technique the single cells are cultured in special kind of apparatus named Cuprak dishes which have two kinds of chambers, small outer chamber filled with water and large inner chamber carrying numerous wells each filled with micro-droplet of medium containing single cells.
(d) Nurse Culture Technique:
In this technique the growth or division of single cell is induced by nurse callus. It has been observed that the single cells plated near the callus tissue divide quickly to give rise to callus tissue. This happening must be due to some leaching effect on the division of single cells by the growing callus mass.
(e) Cell Plating Technique:
The basic technique of plating is to first count the cell number without maceration stage, this will enable a known number of cell units to be established per unit volume of plating media. Both the cell suspension and nutrient medium containing agar are prepared in double concentration separately.
The equal volumes of suspension and the agar medium cooled at 35°C are mixed and then dispersed rapidly into petriplate in such a manner that cells are evenly distributed in a thin layer (~1 mm thick). The dishes are then sealed and incubated for cell division, which will give rise to callus (Fig. 17.4C).
Bergmann (1960) first introduced this most popular technique of plating of cell suspension and this technique is very much useful to calculate the plating efficiency.
The plates may be observed under an inverted microscope and single cells develop into callus, this method ensures the isolation of pure single-cell clones. Usually, plating at cell densities of 103— 105 cells/ml or more yields a high plating efficiency.
Efforts have been made to develop a synthetic medium for cells plated at low density. Cells plated in a culture medium synthesize necessary metabolites and threshold conc. of those helpful for cell division to start. At initial high cell density the equilibrium is reached much earlier than at a low cell density.
It has been observed that below a critical cell density the cells fail to divide. This problem can be overcome by supplementing the minimal medium with some undefined factors like coconut milk, casein hydrolysate or yeast extract and following some different culture techniques.