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Practical 6

The document outlines a practical guide for assessing pollen viability using various staining techniques, including Acetocarmine Staining and the Tetrazolium Test (TTC). It details the materials required and step-by-step procedures for each method, emphasizing the importance of using fresh pollen grains and maintaining proper conditions for accurate results. Additionally, it provides precautions to ensure the reliability of the tests.

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anshikasingh12735
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41 views

Practical 6

The document outlines a practical guide for assessing pollen viability using various staining techniques, including Acetocarmine Staining and the Tetrazolium Test (TTC). It details the materials required and step-by-step procedures for each method, emphasizing the importance of using fresh pollen grains and maintaining proper conditions for accurate results. Additionally, it provides precautions to ensure the reliability of the tests.

Uploaded by

anshikasingh12735
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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Practical: Pollen Viability Test

Aim:
To assess the viability of pollen grains using different staining techniques.

Principle:
Pollen viability is the ability of pollen grains to germinate and successfully fertilize an ovule.
It is an important factor in plant reproduction and breeding. The viability of pollen can be tested
using different methods, such as staining techniques, germination tests, and fluorescence
microscopy. Staining methods, such as the Tetrazolium Test (TTC) and Acetocarmine Staining,
are widely used to determine pollen viability by identifying live and dead pollen grains based
on color changes.

Materials Required:

1. Fresh pollen grains from selected flowers

2. Glass slides

3. Coverslips
4. Acetocarmine stain

5. 2,3,5-Triphenyl Tetrazolium Chloride (TTC) solution

6. Sucrose solution (10-15%)

7. Compound microscope

8. Petri dishes

9. Dropper or pipette

10. Forceps
11. Filter paper

Procedure:

Method 1: Acetocarmine Staining

1. Collect fresh pollen grains from a mature anther.

2. Transfer the pollen grains onto a clean glass slide.

3. Add a drop of 1% acetocarmine solution to the pollen sample.

4. Place a coverslip gently and press lightly to spread the stain.

5. Observe under a compound microscope.


6. Count the number of stained (viable) and unstained (non-viable) pollen grains.
7. Calculate the percentage of viable pollen grains using the formula:
Pollen Viability (%) = (Number of viable pollen grains / Total number of pollen grains) ×
100

Method 2: Tetrazolium Test (TTC Test)

1. Prepare a 1% solution of 2,3,5-Triphenyl Tetrazolium Chloride (TTC) in distilled water.

2. Collect fresh pollen grains and place them on a glass slide.

3. Add a drop of TTC solution and incubate the slide in a petri dish at 25°C for 15-30
minutes.

4. Observe the slide under a microscope.


5. Viable pollen grains will turn red due to enzymatic activity, while non-viable grains
will remain colorless.

6. Calculate the percentage of viable pollen grains.

Method 3: In Vitro Pollen Germination Test

1. Prepare a 10-15% sucrose solution as a germination medium.


2. Collect fresh pollen grains and place them on a petri dish containing the sucrose
solution.
3. Incubate the petri dish at room temperature for 2-4 hours.
4. Observe under a microscope for pollen tube growth.

5. Calculate the percentage of pollen grains showing germination.

Precautions:

1. Use fresh and mature pollen grains for accurate results.


2. Ensure all glassware and slides are clean to avoid contamination.

3. Handle staining solutions carefully to prevent spillage.

4. Maintain the correct temperature and incubation time for TTC and germination tests.

5. Use a fresh sucrose solution for better pollen germination.

6. Observe the samples immediately after staining to avoid drying artifacts.

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