By treating with alchohol and boiling on a water bath.

The correct answer is firstly treating it with Alcohol and allowing it to stand in water bath for a while .So option (D) is correct answer

Separation of intact chloroplast from Green Leaves of angiospermic plant

Introduction:
Chloroplasts are specialized organelles found in plant cells that are responsible for photosynthesis. They contain chlorophyll, which gives plants their green color and plays a crucial role in capturing sunlight for energy production. Separating intact chloroplasts from green leaves is important for various biochemical and physiological studies. Several methods can be used for this purpose, including petrol-ether extraction, chloroform extraction, 10% sucrose solution, and alcohol extraction.

Methods for separation:
1. Petrol-Ether: Petrol-ether extraction is a commonly used method for separating intact chloroplasts from green leaves. The steps involved in this method include:
- Grind the green leaves in a mortar and pestle with a small amount of sand and chilled isolation medium (such as buffer or sucrose solution).
- Filter the homogenate through a muslin cloth to remove large debris.
- Centrifuge the filtrate at a low speed to pelletize the intact chloroplasts.
- Discard the supernatant and resuspend the pellet in a small volume of isolation medium.

2. Chloroform: Chloroform extraction is another method for isolating intact chloroplasts. The steps involved in this method include:
- Grind the green leaves with chilled isolation medium using a mortar and pestle.
- Filter the homogenate through a muslin cloth to remove debris.
- Centrifuge the filtrate at a low speed to pelletize the intact chloroplasts.
- Discard the supernatant and resuspend the pellet in a small volume of isolation medium.

3. 10% Sucrose Solution: Sucrose solution can also be used for the separation of intact chloroplasts. The steps involved in this method include:
- Grind the green leaves with chilled isolation medium containing 10% sucrose using a mortar and pestle.
- Filter the homogenate through a muslin cloth to remove debris.
- Centrifuge the filtrate at a low speed to pelletize the intact chloroplasts.
- Discard the supernatant and resuspend the pellet in a small volume of isolation medium.

4. Alcohol: Alcohol extraction is another method that can be used to separate intact chloroplasts. The steps involved in this method include:
- Grind the green leaves with chilled isolation medium using a mortar and pestle.
- Filter the homogenate through a muslin cloth to remove debris.
- Add alcohol (such as ethanol or methanol) to the filtrate to precipitate the intact chloroplasts.
- Centrifuge the mixture to pelletize the chloroplasts and discard the supernatant.
- Resuspend the pellet in a small volume of isolation medium.

Conclusion:
All of the mentioned methods can be used for the separation of intact chloroplasts from green leaves. The choice of method depends on the specific requirements of the experiment and the accessibility of the reagents. It is important to handle the plant material gently and keep the isolation medium chilled to maintain the integrity of the chloroplasts during the separation process. The isolated intact chloroplasts can then