Model Answer
0 min readIntroduction
Plant tissue culture, a cornerstone of modern botany and biotechnology, relies heavily on the ability to isolate and culture single cells. This is crucial for various applications, including protoplast fusion, genetic transformation, and the creation of haploid lines. The successful establishment of a cell culture begins with the effective isolation of individual cells from intact plant organs, followed by a rigorous assessment of their viability – their capacity to divide and differentiate. This ensures only healthy, functional cells are used for further experimentation and propagation.
Isolation of Single Cells
The process of isolating single cells from plant tissues involves breaking down the tissue structure to release individual cells. This can be achieved through mechanical and enzymatic methods.
Mechanical Isolation
- Maceration: This involves gently crushing or grinding the plant tissue in a suitable medium. The force applied needs to be carefully controlled to avoid cell damage. Commonly used for tissues like leaves and fruits.
- Filtration: Passing the tissue suspension through a series of filters with decreasing pore sizes can separate cells based on size.
- Microdissection: Using fine needles or scalpels under a microscope to manually isolate cells, particularly useful for specific cell types.
Enzymatic Isolation
Enzymatic methods are more commonly used as they offer gentler cell separation.
- Cellulase: Degrades cellulose, the primary component of plant cell walls, releasing individual cells.
- Pectinase: Breaks down pectin, another major cell wall component, aiding in cell separation.
- Macerozyme: A complex enzyme mixture that degrades various cell wall components.
Typically, a combination of these enzymes is used to achieve optimal cell separation. The enzyme concentration, incubation time, and temperature are critical parameters to optimize for each plant species.
Assessing Cell Viability
Determining the viability of isolated cells is essential before initiating culture. Several methods are employed, categorized based on their principles.
Staining Methods
- Evan’s Blue Staining: Evan’s blue dye is excluded by cells with intact membranes. Dead cells with compromised membranes take up the dye and appear blue under a microscope.
- Fluorescein Diacetate (FDA) Staining: FDA is a non-fluorescent dye that is hydrolyzed by esterases present in living cells, producing a fluorescent product (fluorescein). Thus, viable cells fluoresce under UV light.
Membrane Integrity Assays
- Phenol Red Assay: Phenol red changes color (yellow to pink) in response to pH changes. Damaged cells leak intracellular contents, altering the pH of the surrounding medium and causing a color change.
- Electrolyte Leakage: Measures the amount of electrolytes (e.g., potassium, sodium) leaking from cells. Higher leakage indicates compromised membrane integrity and lower viability.
Metabolic Activity Tests
- Tetrazolium (TTC) Reduction Assay: TTC is reduced by active dehydrogenases in living cells to form a red precipitate (formazan). The intensity of the red color is proportional to the metabolic activity and viability of the cells.
- Resazurin Assay: Resazurin is a non-toxic dye that is reduced to resorufin by metabolically active cells, producing a fluorescent pink color.
The choice of viability assay depends on the plant species, the type of tissue, and the specific application. Often, a combination of methods is used to obtain a more accurate assessment of cell viability.
Conclusion
The successful isolation of single cells and accurate assessment of their viability are fundamental steps in plant tissue culture. Mechanical and enzymatic methods, each with its advantages and limitations, are employed for cell isolation. Viability is then determined using a range of techniques, from simple staining methods to sophisticated metabolic assays. Continued refinement of these techniques is crucial for advancing plant biotechnology and crop improvement efforts.
Answer Length
This is a comprehensive model answer for learning purposes and may exceed the word limit. In the exam, always adhere to the prescribed word count.