Experiment 1: Preparation of a Temporary Mount to Observe Pollen Germination

Aim

To study and calculate the percentage of pollen germination on a slide.

Principle

In nature, pollen grains germinate on the compatible stigma of the carpel. This process is called pollen-pistil interaction. It is an essential step in fertilisation of angiosperms which determines compatibility and incompatibility of both pollen and pistil.

This dynamic process involves pollen recognition followed by inhibition or promotion of pollen germination. The pollen grains can be induced to germinate in synthetic medium using chemicals favouring germination such as 10% sucrose solution that facilitates rapid growth of pollen tubes.

During germination, intine (inner wall) of pollen grain emerges as pollen tube through a germ pore in exine (outer wall). The tube grows into genetically compatible stigma only and carries two male gametes inside it.

Out of two male gametes, one fuses with egg cell while other fuses with secondary nucleus. This process is known as double fertilisation, characteristic of all angiosperms. Pollen of different flowers differs in germination time, tube length and wall sculpturing.

Requirements

Mature pollens of any seasonal flower like Tradescantia, jasmine, lily, etc., cavity glass slides, cover slips, microscope, distilled water, beaker, weighing machine, filter paper, glass rod, boric acid, sucrose, calcium nitrate, brushes and dropper.

Procedure

1. Firstly, prepare a pollen germination medium by dissolving 10 g sucrose, 30 mg calcium nitrate and 10 mg boric acid in 100 mL of distilled water. Alternatively, 10% sucrose solution can also be used as the medium.
2. Put a drop of this prepared medium or 10% sucrose solution with the help of a dropper in the cavity slide.
3. Dust the pollen on the medium on same slide with the help of a drawing brush.
4. Now, cover pollens with the cover slip carefully without introducing air bubbles.
5. Observe the slide containing pollen under the microscope after 10–15 minutes.
6. Finally, count the total number of non-germinating pollen and germinating pollen grains in different microscopic fields.

Observation

Count the germinated and total number of pollens per microscopic field and tabulate your readings in the following table. A pollen grain is considered germinated when pollen tube length is greater than diameter of pollen grain.

Calculation

The percentage of pollen germination is calculated by counting germinated pollens relative to total pollens observed. Use accurate counting in at least three to five microscopic fields for reliable results.

Result

All the pollens of different species in the medium germinate with different percentage. Also, there is great variation in time taken for the germination of pollen of each species. Pollen viability and germination rate depend on species, medium concentration and environmental conditions.

Precautions

• The nutrient solution should be made carefully otherwise, pollen grains might fail to germinate.
• Only few pollen grains should be dusted in the nutrient solution in order to avoid overloading or overcrowding.
• While placing cover slip, air bubbles should be avoided else they might interfere with observation and counting of pollens.
• The entire process should be carried at room temperature (25 ± 2 °C).
• Use freshly collected, mature and viable pollen grains and clean, dry cavity slides for accurate observation.