The below mentioned article provides a study notes on Single Cell Clone.
By culturing a single isolated cell single cell clone can be obtained. Single cell clones derived from callus or suspension culture are heterogenous. They differ from one another in shape, texture, friability, colour, growth and morphogenetic potential etc.
Working with Nicotiana tabacum L. Winsconsin (’38), Murashige and Nakamo (’65) reported that single cell clones differ in their organ forming potential. Vasil and Hildebrandt (’65) working with hybrid tobacco also observed that single cell clones derived from a newly initiated culture differ in their capacity to produce plant- lets by shoot-bud initiation.
On continuing subculture single cell clones which were initially totipotent lost this capacity.
Single cell clones can be obtained usually by three methods.
These are:
(a) Paper raft nurse technique,
(b) Petridish plating technique, and
(c) Growth of isolated cells in a micro-chamber.
(a) Paper Raft Nurse Technique:
This technique was developed by Muir (’53). Isolated cells are placed on a filter paper raft placed over an actively growing nurse culture. This nurse culture supplies the cell clones all essential nutrients required for growth and also the factor needed to induce division through the paper barrier.
(b) Petridish Plating Technique:
Bergman (’60) first plated out cell suspension on agar plates. In this method the filtrate from suspension culture is subjected to cellular unit count. The counted suspension is adjusted either by diluting or concentrating by low speed centrifugation.
Sterile medium is prepared with 0.6% agar and cooled at 35°C. In this sterile medium 2 ml of suspension culture of known cellular density is mixed while the agar is still warm. Then the cells are immediately plated as a thin layer in a sterile petridish, which is sealed with parafilm. Cellular units can be counted with a steriobinocular microscope. From this the number of cellular unit per square milimitre per plate can be calculated.
The plates are then incubated at 25°C for 21 days. The cells divide and form colonies. The rate of cell division depends upon the density of cells per plate. In a plated culture cell division first occurs at cell aggregate region and then in free cells.
Various factors influence the rate of cell division in culture. Increase in carbon dioxide concentration in the environment increases cell division. Ethylene at low concentration (2.5 mg/l) helps growth. A balance between auxin and cytokinin influences cell division.
The number of colonies per plate can be determined by shadow graph prints. The plating efficiency (PE) can be calculated from the following equation.
PE = Number of colonies per plate × 100 Number of cellular units per plate
Plating efficiency can be increased by increasing the iron concentration or adjusting the pH.
In low density plate culture of Convolvulus cells presence of cytokinin, amino acids and proper concentration of auxin helps good growth. At low plating density sycamore cells need a medium containing cytokinin, gibberellin and amino acids. But these are not required for normal growth.
(c) Growth of Isolated Cells in a Micro-Chamber:
De Ropp (’55) first attempted to culture single cells in hanging drop of a medium in a micro-chamber. Torrey (’57) used Maximov’s double covership method for culturing single cells from pea root. He placed at the centre of the agar medium a piece of parent callus tissue and the cells were placed around the outer part of the agar medium.
The parent callus tissue is supposed to nurse the isolated cells. After several weeks 8% cells show division.
Jones, Hildebrandt, Riker and Wu (’60) used micro-chamber sealed with inert mineral oil. They cultured callus cells from a hybrid of Nicotiana tabacum x N. glutinosa. Cell division was noted when there were at least thirty cells in the culture.
Significance:
(1) By single cell clone culture resistant clones can be selected. By this method Carlson (’73) obtained cells resistant to methionine sulfoximine.
(2) By single cell clone culture we can obtain valuable information about cell physiology.