Let us make an in-depth study of the meaning, principle, factors affecting and importance of single cell culture.
What is the Meaning of Single Cell Culture?
Single cell culture is a method of growing isolated single cell aseptically on a nutrient medium under controlled condition.
Principle of Single Cell Culture:
The basic principle of single cell culture is the isolation of large number of intact living cells and cultures them on a suitable nutrient medium for their requisite growth and development. Single cells can be isolated from a variety of tissue and organ of green plant as well as from callus tissue and cell suspension. Single cells from the intact plant tissue (leaf, stem, root cladode etc.) are isolated either mechanically or enzymatically.
Mechanical isolation involves tearing or chopping the surface sterilized explant to expose the cells followed by scraping of the cells with a fine scalpel to liberate the single cells hoping that it remains undamaged. But very few living cells are obtained for a lot of time and effort. Gentle grinding of surface sterilized explant in a sterilized mortar-pestle followed by cleaning the cells by filtration and centrifugation is now widely used for the large-scale mechanical isolation of viable cells.
A considerably more efficient way of large-scale isolation of free cells from the surface sterilized is to dissolve the intercellular cementing material, i.e. pectin, by pectinase or macerozyme treatment. The enzyme macerates the tissue from which large-number of variable cells can be obtained. The special feature of enzymatic isolation of cell is that it has been possible to obtain pure preparation of viable cells with less effort and time.
The single cells are traditionally isolated from the established friable callus tissue and cell suspension culture. Mechanically, single cells are carefully isolated from cell suspension or friable callus with a needle or fine glass capillary. Alternatively, the friable tissue is transferred to liquid medium and the medium is continuously agitated by a shaker.
Agitation of liquid medium breaks and dispenses the single cells and cell clumps in the medium. As a result, it makes a cell suspension. The cell suspension is first filtered to remove cell clumps and the filtrate is then centrifuged to collect the single cells from the pellete.
The isolated single cell can be cultured either in liquid medium or on solid medium. There are five basic methods that are used for culturing single cells such as paper raft nurse technique the petri dish plating technique, the micro-chamber technique, the micro-droplet technique, the plating with nurse tissue technique. In culture, the single cells divide re-divide to form a callus tissue. Such callus tissue also retains the capacity to regenerate the plantlets through organogenesis and embryogenesis.
Factors Affecting Single Cell Culture:
1. The composition of the medium for the growth of single cell culture is generally more complex than callus and cell suspension culture. For example, Convolvulus cells require a cytokinin and amino acids that are not necessary for the callus culture of that species.
2. Induction of division of single cells using paper raft technique indicates that isolated cells get the exact essential nutrient from the callus mass. It has been suggested that the callus mass leaches out the essential nutrient through plasma membrane of the cells.
3. In case of petri dish plating technique the initial plating cell density is very critical.
Importance of Single Cell Culture:
Single cell culture technique is very important for the fundamental and mutation studies and it has a wide industrial application.
1. Single cell culture could be used successfully to obtain single cell clones.
2. Plants could be regenerated from the callus tissue derived from the single cell clones (Fig 9.6).
3. The occurrence of high degree of spontaneous variability in the cultured tissue and their exploitation through single cell culture are very important in relation to crop improvement programmes.
4. One of the major problems of mutation breeding in higher plants is the formation of chimeras following the mutagenic treatment of multi-cellular organism. In this respect single cell culture method are more efficient. Isolated single cells can be handled as a microbial system for the treatment of mutagens and for mutant selection.
In practice, single cells are grown on a medium containing the mutagenic compounds and the proliferating cell lines are isolated. The mutant nature of the selected cell lines can be confirmed by regenerating the plants and comparing their phenotypes with a normal plant. Many cell lines resistant to ammo acid analogues, antibiotics, herbicides, fungal toxins etc. have been selected by this simplest method.
5. Many plants synthesize various important natural compounds in the form of alkaloids, steroids etc. Some of these natural compounds are highly medicinally important. Several workers have reported the synthesis of several times higher amounts of alkaloid by cell culture than the alkaloid content in the intact plant. So, from the commercial point of view, single cell culture in large-scale could become a valuable technique for industrial production of such important natural compound.
6. Biotransformation means the cellular conversion of an exogenously supplied substrate compound not available in the cell or the precursor of a particular cellular compound to a new compound or the known compounds in higher amounts.
A cell can be described as a metabolic factory where a large-number of enzyme systems are working. When the cells are fed with analogues or intermediate or precursor compounds of a metabolic pathway, the cells place them immediately to the particular metabolic pathway and switch on its machinery for the production the Particular compound. Single cell culture is an ideal system for the study of biotransformation.
Generally, two approaches are now being followed for biotransformation studies using single cell culture such as:
(i) Cells are fed with substrate compounds normally not available to the plant. The main objective of such feeding is to obtain a new compound through the biotransformation process.
(ii) Cells are fed with precursor of a compound available in the plant. The main objective of such feeding is to enhance the production of a compound.
7. Induction of polyploidy has been found to be very useful for plant breeding to overcome the problem of sterility associated with hybrids of unrelated plants. Polyploidy can easily be achieved by single cell culture.
A large-number of genetically sterile hybrids exist in the genus Saccharum. When cell culture of such sterile hybrid is treated with 50 mg/L colchicine for 4 days, it has been found that about 48% of such treated cells become uniformly polyploid. These polyploid cells are then induced to regenerate a large-number of fertile plants. In this regard, cell culture is very useful with other crops also.