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Suspension Culture, , The culture and maintenance of individual plant cells, in a liquid medium is known as suspension culture or. cell, . culture.
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The simple procedure for suspension Culture 1s given, below:, , 1. Callus is cultured from a desired explant source,, , 2. The well grown callus is transferred to liquid MS, medium containing 2,4-D (mg/l) and sucrose (750 mg/l) in, a flask. “, 3. The culture flask is kept on a rotary shaker and, agitations of 100 rpm is set up in the shaker Sysiem. The, shaker is placed in a constant temperature room at 2IC,, , 4, The agitations break the callus into individual cells., Within 2-3 weeks, sufficient number of individual cells, develop in the medium., , 5. A small amount of the cell suspension is transferred, to fresh medium for sub-culturing., , 6. Sometimes, the cells are plated on semi-solid MS, medium and incubated at 25°C for a few weeks to induce, embryoid formation. Plantlets are regenerated from the, embryoids. ; ,, , Methods For Growth Measurement, , The growth rate of cells in suspension culture is measured in the folowing ways: _, , 1. Cell packing method., , 2. Direct cell count method., , 3. Direct weight method., , 4. Protein measurement technique., , 1. Cell Packing Method: A 10ml of culture, suspension is transferred to a graduated measuring tube and, centrifuged for a few minutes. The cells get settled at the, bottom of the tube. The total volume of packed cells is, measured against the readings of the graduated measuring tube., Then the concentration of cells in the culture is expressed in, percentage to the total volume of the suspension.
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Measuring tube conta, Centrifugation Measurement of packed, ining cell suspension, , cell volume in measuring tube, , Fig. 20.13: Measurement of cell growth in suspension, culture using cell packing method., , 2. Direct Cell Count Method: A drop of cell, suspension is placed on a haemocytometer. The latter is viewed, through a microscope. The number of cells is counted and, calculated with reference to the total volume of the suspension., , Cell clumps, if any in the suspension cause difficulties, in cell counting. The cell clumps are made to release the, _cells by treating with chromic trioxide or pectinase., , 3. Direct Weight Method: A known volume of cell, suspension is filtered through a whatmann nn.] filter. The, , filter is dried and weighed. Its original weight i is reduced, , from the final weight. The weight of cells is expressed in, grams/litre., , 4. Protein Measurement Technique: Protein is, extracted from a known volume of cell suspension, and is, , measured by using Lowry’s method. The protein contents i is, expressed in mg/ml., , The protein contents of the filtrate should not exceed, | mg/ml. It indicates the maximum concentration of cells in, the suspension. Immediately the suspension is sub-cultured, , for further use.
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Experiments to Assess the Cell Viability, 1. Direct Visual Test : The viability of cells cay be, determined by direct observation of cells under a microseg, , The living cells show protoplasmic streaming movement and, active functional nucleus. But the dead cells do not have any, , protoplasmic streaming movement., , 2. Emans Blue Dye Treatment : The cell suspen., sion is treated with Eman’s blue dye at 0.025 wyy, concentration. The dead cells take the dye from the medium,, and become blue in colour. But living cells exclude the dye, so that they remain green. The culture containing more, number of green cells is used for sub-culturing., , 3. Chemical Method: When the cells are treateq, with fluorescein diacetate (0.01% solution), it binds with the, cell’s surface. After five minutes, the cells are observed under, a fluroscence microscope. The living cells emit fluroscent, radiations and appear green. It indicates living cells in the, suspension., , Uses of Suspension Culture, , 1. Suspension cultures are used as model systems to, study metabolic pathways in plant cells., , 2. They are used to regenerate identical clones from, single cells. :, , 3. They are used in invitro mutation studies:, , 4. They are used to isolate plant protoplasts for gene, manipulation., , 5. They are used as sources for extraction of cellular, enzymes and many secondary metabolites.
