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Testing the Viability of Isolated Protoplasts Introduction Isolated protoplasts must be tested for viability to ensure they are alive and functional for experimental purposes. Viability confirms the integrity of the protoplast membrane and the presence of active metabolic processes necessary for further studies like culture, hybridization, or genetic transformation.   Methods for Testing Viability Fluorescent Dye Staining: Fluorescein Diacetate (FDA): FDA is a non-fluorescent dye that enters protoplasts and is converted into a green fluorescent compound by active enzymes in viable cells. Observed under a fluorescence microscope. Propidium Iodide (PI): PI stains only dead cells red as it penetrates damaged membranes, while viable cells exclude the dye. Evans Blue Staining: Dead cells absorb the blue dye, while viable protoplasts remain unstained. Simple ...

Protoplast Isolation: Methods Protoplasts are isolated using two primary methods: mechanical and enzymatic . 1. Mechanical Method This method is suitable for isolating protoplasts from large, highly vacuolated cells, such as those in storage tissues like onion bulb scales, radish roots, and beet roots. Steps: 1.         Plasmolysis: o    Cells are treated with an isoosmotic solution, causing the cytoplasm to shrink and move to the center of the cell. 2.         Dissection: o    The tissue is finely dissected to release the protoplasts. 3.         Deplasmolysis: o    The dissected tissue is transferred to water or another solution, allowing the protoplasts to swell and release. Historical Note: ·         Klercker (1892) was the first to isolate protoplasts mechanically from Stratiotes aloides .   2. Enzymati...

Definition: Somatic hybridization is a biotechnological technique in which two different somatic cells (non-reproductive cells) from different species or varieties are fused to form a hybrid cell. This process allows the combination of genetic material from two distinct sources to create a novel hybrid organism. How It Works: Somatic hybridization is achieved through protoplast fusion , where the cell wall is removed, and only the protoplasts (cell contents enclosed by the plasma membrane) are fused. Steps Involved in Somatic Hybridization: Isolation of Protoplasts: Plant cells are treated with enzymes like cellulase and pectinase to remove the cell wall. Fusion of Protoplasts: Fusion is induced using chemicals (e.g., polyethylene glycol - PEG) or electrical stimulation (electrofusion). Selection of Hybrid Cells: Only fused cells are selected based on their growth on selective media. Regeneration into Plants: The hybrid protoplasts are cultured to regenerate a complete plant using tiss...

Introduction: Cryptococcosis is a fungal infection caused by Cryptococcus neoformans or Cryptococcus gattii . It primarily affects the lungs and can spread to the central nervous system (CNS) , leading to meningitis . This infection is more common in individuals with weakened immune systems, especially those with HIV/AIDS . Causes and Transmission: Causative Agent: Cryptococcus neoformans : Found in soil contaminated with pigeon droppings . Cryptococcus gattii : Found in certain trees and decaying wood. Mode of Transmission: Inhalation of fungal spores: From soil or contaminated environments. Not spread from person-to-person. Types of Cryptococcosis: Pulmonary Cryptococcosis: Infection in the lungs. CNS Cryptococcosis (Cryptococcal Meningitis): Most severe form; infection spreads to the brain and spinal cord. Disseminated Cryptococcosis: The fungus spreads to other parts of the body (e.g., skin, bones). Symptoms: Pulmonary Cryptococcosis: Cough Chest pain Shortness of breath Fever...