Methods of Single Cell Isolation
Methods of Single Cell Isolation
A) From Plant Organ:
The most material for the isolation of single cell aseptically on a nutrient medium under controlled conditions.
Method of Single Cell Isolation:
A) From Plant Organ:
The most suitable material for the isolation of single cells is the leaf tissue since a more or less homogeneous population of cells in the leaves offer good candidates for raising defined and controlled large scale cell culture. From such intact plant organs single cells can be isolated using mechanical or enzymatic methods.
i) Mechanical Method:
Mechanical isolation involves tearing or chopping surface sterilized explant to expose the cells followed by scrapping of the cells with a fine scalpel to liberate the single cells hoping that it remained undamaged. Gnanam and Kulandaivelu ( 1969) developed a procedure to isolate mesophyll cells, active in photosynthesis and respiration, from mature leaves of several species of dicot and monocot. The procedure involves mild maceration of 10g leaves in 40 ml of grinding medium (20 µ mol sucrose, 10µ mol Mgcl2, 20 µ mol tris –HCL buffer, Ph7.8) with a mortar and pestle. The homogenate is passed through two layer of muslin cloth and cell thus released are washed by centrifugation at low speed using the same medium.
The mechanical isolation of free parenchymatous cells can also be achieved on a large scale.
ii) Enzyme Method:
Takebe et.al. (1968) treated tobacco leaf tissue with enzyme pectinase and obtained a large number of metabolically active cells. The potassium dextran sulphate in the enzyme mixture improved the yield of free cells.
Isolation of single cells by the enzymatic method has been found convenient, as it is possible to obtain high yields from preparation of spongy parenchyma with minimum damage or injury to the cells. This can be accomplished by providing osmotic protection to the cells while the enzyme macerozyme degrades the middle lamella and cell wall of the pranchymatous tissue. Applying enzymatic method to cereals (Hordeum vulgare, Zea mays) has proven rather difficult since the mesophyll cells of these plants are apparently elongated with number of interlocking constrictions, thereby preventing their isolation.
B) From Cultured Tissue:
The most widely applied approach is to obtain a single cell system from cultured tissue. Freshly cut piece from surface sterilized plant organs are simply placed on a nutrient medium consisting of a suitable proportion of auxins and cytokinins to initiate cultures. Explant on such a medium exhibit callusing at the cut ends, which gradually extends to the entire surface of the tissue. The callus is separated from an explant and transferred to a fresh medium of the same composition to enable it to built up a mass tissue. Repeated sub-culture on an agar medium improves the friability of the callus, a pre-requsite for raising a fine cell suspension in a liquid medium. The piece of undifferentiated and friable callus are transferred in a continuously agitated liquid medium consisting of flask or suitable vials. Agitation is done by placing the culture medium exerts mild pressure on small pieces of tissue, breaking them into free cells and small cell aggregates. Further, it augments the gases exchange between the culture medium and the culture air and also ensures uniform distribution of cells as wells as clumps in the medium.