Breeding and Seed Production of Ornamental Crops (2+1)

Exercise - 3

Aim: Determining the pollen viability through staining tests.
Objectives: To assess the potential of a given pollen parent to serve as a pollinizer.
Materials required:
Microscope, microscopic and cavity slides water glasses, cover slips, incubator, burner, stains, reagent bottles, distilled water, measuring cylinder, beakers, scissors, forceps, dessicator, glass vials, muslin cloth, refrigerator, pollen source.
Staining Tests:
1. ACETO/PROPIONO CARMINE (1-2%)TEST
Weigh 1 or 2 g of carmine powder, dissolve it in 95ml of glacial acetic/propionic acid. Add distilled water to make a total of 100ml solution. Boil it, cool and filter. Add few drops of 10% ferric chloride solution (10 g ferric chloride dissolved in distilled water to make a final volume of 100ml), mix it and store in a refrigerator.
Testing:
Shed the freshly collected pollen on a clean slide and put a drop or two of freshly prepared stain over it. Agitate the pollen grains with a needle for about 30 seconds. Place the cover slip, held in forceps, gently to avoid air bubbles. After a few minutes, examine the slide under a microscope. Coloured pollen grains are considered viable. Take 2-3 readings, avoids taking observations towards the edges of cover slip. Calculate average pollen viability percentage.
2. IODINE(0.5%)TEST
Preparation
Dissolve 500mg each of potassium iodide and iodine in distilled water to make a final volume of 100ml.
Procedure
Pour a little amount of pollen on a clean side. Put 1 or 2 drops of the dye over pollen and mix thoroughly. Place a cover slip and count the number of darkly stained (viable) pollen grains under the microscope. Take reading for several times and work out the average to give percent pollen viability.
3. Erythrosin B (0.04%) TEST
Preparation
Dissolve 40mg of erythrocin B in distilled water to make a final solution of 100ml.
Procedure
Follow the staining procedure as done in carmine/iodine test. Count the number of unstained pollen grains, which are considered viable in this method, under a microscope. Calculate percent pollen viability.
4. TETRAZOLIUM (MTT) TEST
Preparation
Dissolve 10g of 3(4,5-dimethyl thiazolyl 1-2) 2,5- diphenyl tetrazolium bromide (MTT) in distilled water to make a final solution of 100ml. Prior to use, dilute it at the rate of 1 part stain to 10 parts of 60% sucrose.
Staining procedure
Dust pollen on a neat and clean slide. Add a drop of stain and then place a cover slip. Keep the slide for 30-60 minutes at 30-57^oC. Count light red or red pollen as viable and colourless or deep pollen as non viable. Calculate the viability percentage.
Precautions:

1. Good sanitation should be maintained in and around the area
2. Use gloves while handling the chemicals.
3. Freshly prepared stains should be used.
4. Sterilized instruments should be used.