Class 12 Biology Notes Chapter 3 (Chapter 3) – Lab Manual (English) Book

Detailed Notes with MCQs of Experiment number 3 from your Lab Manual, which deals with Pollen Germination. This is a fundamental process in plant reproduction and understanding the experiment is important, not just for your practical exams, but also for conceptual clarity needed in competitive government exams.

Experiment 3: Study of Pollen Germination on a Slide

1. Aim:
To observe and study the process of pollen germination on a glass slide using an artificial nutrient medium.

2. Principle:
Pollen grains represent the male gametophyte in flowering plants. When a mature pollen grain lands on a compatible stigma (in vivo), it germinates. This involves the emergence of a pollen tube from one of the germ pores. The pollen tube grows down through the style, carrying the male gametes to the ovule for fertilization.
This germination process requires specific conditions: moisture, suitable temperature, and nutrients. We can simulate these conditions in vitro (outside the living organism, on a slide) using a nutrient solution containing sugars (like sucrose) and minerals (like boron). The pollen grain absorbs this solution, becomes metabolically active, and the vegetative cell (tube cell) elongates to form the pollen tube, which emerges through a germ pore.

3. Materials Required:

• Biological Material: Freshly plucked, mature flowers with easily available pollen (e.g., Vinca rosea (Sadabahar), Hibiscus rosa-sinensis (China rose), Petunia, Balsam, Crotalaria, Tecoma). Ensure flowers are freshly opened.
• Chemicals:
  • Sucrose (Sugar)
  • Boric Acid (Boron source)
  • Distilled Water
• Glassware & Equipment:
  • Glass slides
  • Coverslips
  • Beaker
  • Dropper
  • Petri dish (to create a moist chamber)
  • Compound Microscope
  • Brush (fine-tipped) or needle
  • Filter paper

4. Procedure:
* Preparation of Nutrient Medium: Prepare a 10% sucrose solution by dissolving 10g of sucrose in 100ml of distilled water. To this, add a small pinch (about 10-20 mg or 10-20 ppm) of boric acid. Stir well to dissolve. (Note: The optimal concentration may vary slightly depending on the plant species).
* Slide Preparation: Take a clean, grease-free glass slide. Place one or two drops of the prepared nutrient medium in the center using a dropper.
* Pollen Collection: Select a fresh flower. Gently dust the pollen grains from its mature anthers onto the drop of nutrient medium on the slide. This can be done by tapping the anther over the slide or using a clean brush to transfer the pollen. Avoid taking too much pollen; it should be sparsely distributed.
* Mounting: Carefully lower a clean coverslip onto the drop, avoiding air bubbles. You can place the edge of the coverslip first and gently lower it using a needle.
* Incubation: Place the slide in a moist chamber. A simple moist chamber can be made by placing the slide in a petri dish lined with moist filter paper or cotton wool. This prevents the nutrient medium from drying out. Leave it undisturbed for about 15-30 minutes at room temperature.
* Observation:
* First, observe the slide under the low power (10X objective) of the compound microscope. Focus on the pollen grains. Note their initial appearance.
* After the incubation period (15-30 mins), observe the slide again under low power. Look for signs of germination – the emergence of pollen tubes.
* Switch to high power (40X or 45X objective) for a detailed view of the germinating pollen grains and the growing pollen tubes. Observe the cytoplasm and potentially the nuclei within the tube.

5. Observations:
* Initially, you will see numerous pollen grains scattered in the nutrient medium. Note their specific shape, size, and the texture of the outer wall (exine).
* After incubation, some (or many, depending on viability and conditions) pollen grains will show a thin, tubular outgrowth – the pollen tube – emerging from one of the germ pores.
* Record the approximate percentage of pollen grains that have germinated.
* Observe the structure of the pollen tube; it contains cytoplasm, a tube nucleus (vegetative nucleus), and a generative cell (which may divide into two male gametes, though this might be difficult to observe clearly).
* Draw labelled diagrams of:
* A pollen grain before germination.
* A pollen grain showing germination and pollen tube growth.

6. Results and Conclusion:
* Pollen grains successfully germinate in the prepared nutrient medium on the glass slide, as evidenced by the development of pollen tubes.
* The nutrient medium (containing sucrose and boric acid) provides the necessary requirements for in vitro pollen germination and initial pollen tube growth.

7. Precautions:
* Always use freshly opened flowers for viable pollen.
* The glass slide and coverslip must be clean and dry before use.
* Avoid using excess pollen grains on the slide to prevent clumping.
* Ensure the nutrient medium drop does not dry out; use a moist chamber during incubation.
* Place the coverslip gently to avoid trapping air bubbles, which can obstruct observation.
* Handle the slide carefully, especially when placing it on or removing it from the microscope stage.

8. Key Concepts for Government Exams:

• Pollen Grain: Male gametophyte, typically spherical or oval. Has two layers:
  • Exine: Tough outer layer made of sporopollenin (highly resistant to chemical and biological degradation). It may have patterns/sculpturing specific to the species. It has apertures called germ pores where sporopollenin is thin or absent.
  • Intine: Thin inner layer made of cellulose and pectin.
  • Contains cytoplasm, a large vegetative cell (tube cell) with its nucleus, and a smaller generative cell (which floats in the cytoplasm of the vegetative cell). The generative cell divides mitotically to form two non-motile male gametes either before pollen shedding (in ~60% angiosperms) or within the pollen tube after germination.
• Pollen Germination: The process where the intine, covered by the vegetative cell's cytoplasm, emerges through a germ pore to form the pollen tube. Driven by the vegetative/tube nucleus.
• Nutrient Medium Role:
  • Sucrose: Acts as a respiratory substrate (energy source) and helps maintain the osmotic pressure of the medium, preventing the pollen tube from bursting.
  • Boric Acid (Boron): Crucial for pollen germination and pollen tube elongation. Boron is involved in cell wall metabolism (pectin synthesis), sugar transport, and membrane integrity. Its deficiency severely inhibits pollen tube growth.
  • Calcium ions (sometimes added): Also play a role in pollen tube growth and directional guidance.
• In vitro vs In vivo: In vitro germination (on slide) tests pollen viability and basic requirements. In vivo germination (on stigma) involves complex pollen-pistil interaction, including recognition, hydration, and chemical signalling between the pollen and the stigma/style.
• Pollen Viability: The duration for which pollen grains remain functional (capable of germination). Highly variable (e.g., 30 minutes in cereals like rice and wheat, several months in some Rosaceae, Leguminosae). Can be affected by temperature and humidity.
• Significance: Understanding pollen germination is vital for plant breeding (hybridization), assessing pollen viability, studying reproductive barriers, and improving crop yields.

Multiple Choice Questions (MCQs):

1. In the experiment to study pollen germination, sucrose is added to the nutrient medium primarily to:
   a) Provide structural support to the pollen tube.
   b) Act as an energy source and maintain osmotic balance.
   c) Stimulate the division of the generative cell.
   d) Make the exine permeable to water.

2. Which element is critically required in the nutrient medium for proper pollen tube elongation?
   a) Nitrogen
   b) Phosphorus
   c) Boron
   d) Potassium

3. The pollen tube emerges from the pollen grain through an aperture called the:
   a) Stomium
   b) Micropyle
   c) Germ pore
   d) Lenticel

4. The tough, resistant outer layer of the pollen grain, composed of sporopollenin, is known as the:
   a) Intine
   b) Exine
   c) Tapetum
   d) Endothecium

5. During in vitro pollen germination, the pollen tube develops from the:
   a) Generative cell
   b) Male gamete
   c) Vegetative cell (Tube cell)
   d) Exine layer

6. Which of the following is a necessary precaution while setting up the pollen germination experiment?
   a) Using pollen from dried flowers.
   b) Allowing the nutrient medium to dry slightly for better observation.
   c) Overcrowding the slide with pollen grains.
   d) Using a moist chamber during incubation.

7. Sporopollenin, found in the pollen exine, is:
   a) A protein easily digested by enzymes.
   b) A lipid that stores food.
   c) One of the most resistant organic materials known.
   d) A carbohydrate involved in cell wall structure.

8. The function of the pollen tube is to:
   a) Protect the pollen grain from desiccation.
   b) Carry the male gametes to the ovule.
   c) Absorb nutrients from the style.
   d) Produce hormones for fruit development.

9. Observing pollen germination in vitro differs from in vivo germination mainly because in vivo germination involves:
   a) Requirement of Boron
   b) Formation of a pollen tube
   c) Pollen-pistil interaction and recognition
   d) Division of generative cell

10. If you observe a pollen grain under the microscope after successful germination in the nutrient medium, you would expect to see:
    a) Only the exine and intine layers.
    b) The pollen grain with an emerging pollen tube containing cytoplasm.
    c) Two distinct pollen grains fused together.
    d) The pollen grain shrinking due to water loss.

Answer Key for MCQs:

1. b
2. c
3. c
4. b
5. c
6. d
7. c
8. b
9. c
10. b

Study these notes thoroughly. Understanding the 'why' behind each step and component is crucial for competitive exams. Let me know if any part needs further clarification.