Single Cell Culture: Meaning, Principle, Factors and Importance | Plant Tissue Culture

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.