Describe the enzymatic method of isolation of protoplasts taking the example of Carrot.

Enzymatic Isolation of Protoplasts from Carrot

The enzymatic method for protoplast isolation involves several key steps, each designed to maximize protoplast yield and viability. The following details the procedure using carrot as an example:

1. Selection of Plant Material and Preparation

Young, actively growing carrot root tissues are preferred as they contain thinner cell walls and higher enzyme accessibility. The root is surface sterilized by washing with 70% ethanol for 30 seconds, followed by 0.1% mercuric chloride for 5-10 minutes, and finally rinsing thoroughly with sterile distilled water 3-5 times. This sterilization prevents contamination during the isolation process.

2. Cell Wall Degrading Enzyme Cocktail Preparation

A mixture of cell wall degrading enzymes is crucial for efficient protoplast release. The most commonly used enzymes include:

• Cellulase: Degrades cellulose, the major component of the plant cell wall. (e.g., Onozuka Cellulase R-10)
• Pectinase: Degrades pectin, another significant cell wall component. (e.g., Pectolyase Y-23)
• Macerozyme: A complex enzyme preparation that degrades various cell wall polysaccharides.

The optimal concentration of each enzyme varies depending on the plant species and tissue type. For carrot, a typical enzyme cocktail might contain 1-2% Cellulase R-10, 0.5-1% Pectolyase Y-23, and 0.1-0.2% Macerozyme in a suitable buffer solution (e.g., mannitol solution).

3. Incubation with Enzymes

Sterile carrot tissue segments (approximately 1 mm³) are incubated in the enzyme solution in a Petri dish or flask. The incubation is typically carried out in the dark at 25-30°C with gentle shaking (60-80 rpm) for 1-3 hours. The duration of incubation needs optimization; prolonged incubation can lead to protoplast damage, while insufficient incubation results in incomplete cell wall degradation.

4. Osmotic Stabilization and Protoplast Release

During incubation, the cells are often subjected to osmotic stress due to the enzyme solution. To prevent protoplast bursting, a suitable osmoticum, such as mannitol or sorbitol (1-1.5 M), is added to the enzyme solution. This maintains the osmotic potential and prevents water influx into the protoplasts. After incubation, the tissue is gently stirred or agitated to facilitate protoplast release.

5. Protoplast Purification and Isolation

The released protoplasts are separated from undigested tissues and enzyme debris using a series of filtration steps:

• Coarse Filtration: Using a nylon mesh (e.g., 200 µm) to remove large tissue fragments.
• Fine Filtration: Using a nylon mesh (e.g., 80 µm) to further purify the protoplast suspension.
• Centrifugation: Protoplasts are pelleted by gentle centrifugation (100-150 g for 5-10 minutes) in a suitable buffer.

The protoplast pellet is then gently resuspended in fresh osmoticum solution for further use or analysis.

6. Assessing Protoplast Viability

Protoplast viability is crucial for downstream applications. Common methods to assess viability include:

• Evan’s Blue Staining: Dead protoplasts with damaged membranes take up the dye, while viable protoplasts remain unstained.
• Fluorescein Diacetate (FDA) Staining: FDA is hydrolyzed by esterases in viable protoplasts, producing a fluorescent product.
• Microscopic Observation: Observing protoplast morphology and cytoplasmic streaming under a microscope.