Single cell culture

single cell culture

1. Single Cell Culture Mrs. Praveen Garg VITS College, satna

2. Introduction Single cell culture is a method of growing isolated single cell aseptically on a nutrient medium under controlled condition. The single cells can be isolated from a variety of tissue and organ of intact plant or from callus tissue or from cell suspension by using either mechanical method or chemical method. 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. The basic principle of single cell culture is the isolation of large number of living cells and cultures them on a suitable nutrient medium for their requisite growth and development.

3. Methods of Isolation In 1902, Haberlandt was the first person who made pioneering attempts to isolate and culture a single cell from a flowering plant.  There are two methods of isolation . Mechanical isolation: It 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 . Chemical isolation: I n this method, the macerozyme or pectinase can be used to dissolve the middle lamella and releasing single cells from intact tissue.

4. Development of Plant cells from a single cell culture in M icrochamber

5. The single cells are 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 .

6. There are various methods of single cell culture. Paper raft nurse culture technique Micro- chamber technique Micro- droplet technique Nurse culture technique Cell plating technique.

7. 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. 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. Micro-Chamber Technique

8.  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. 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.

9. 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. The dishes are then sealed and incubated for cell division, which will give rise to callus.  

10. 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 10 3 — 10 5  cells/ml or more yields a high plating efficiency. O bservation

11. Factors Affecting Single Cell Culture The composition of the medium for the growth of single cell culture is generally more complex than callus and cell suspension culture. 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. In case of petri dish plating technique the initial plating cell density is very critical.

12. 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. Single cell culture could be used successfully to obtain single cell clones. Plants could be regenerated from the callus tissue derived from the single cell clones.   Single cell culture are very important in relation to crop improvement programmes . Isolated single cells can be handled as a microbial system for the treatment of mutagens and for mutant selection. Many plants synthesize various important natural compounds in the form of alkaloids, steroids etc. Some of these natural compounds are highly medicinally important. Single cell culture is an ideal system for the study of biotransformation .

13. THANK YOU