Pollen Germination and Pollen Viability (PROTOCOLS)

In vitro pollen germination

It can be assessed with hanging drop method. Pollen germination and pollen tube growth can be
determined by placing a small drop of germination medium on a cover glass; pollen grains are
speckled on that drop with a clean brush, and cover glass are then inverted and rested on cavity
slide. Pollen are incubated under dark conditions at 25°C in a culture medium containing 5%
sucrose, 5 ppm boric acid (H3BO3), and 1% agar for 24, 48, and 72 hr. For each incubation
period, germination is recorded in 3 drops by counting 3 fields of microscope.

A pollen grain is considered germinated when pollen tube length is at least equal to or greater
than grain diameter. Germination percentage (%) is determined by dividing number of
germinated pollen grains per field of view by total number of pollens per field of view.
Measurements of pollen tube length (μm) are recorded directly by an ocular micrometer fitted to
eyepiece of the microscope. Mean pollen tube length is calculated as average length of 5 pollen
tubes measured from each drop.
Pollen viability test

Pollen viability can be estimated using 2 colorimetric tests, using 2, 3, 5 triphenyl tetrazolium
chloride (TTC) or acetocarmine. Both methods allow addition of colorant on pollens, followed by
observation under a compound microscope. Pollen viability can be scored according to staining
level (pollen with bold red colour as viable and colourless pollen as nonviable).

Pollen viability = (Number of viable pollen grains / Total number of pollen grains) x 100

Pollens stained with TTC and photographed using light microscopy.

Area of each square = 1 mm2; Depth of chamber = 0.1 mm;
Volume = depth x area = 0.1 mm x 1 mm2 = 0.1 mm3
0.1 mm3 = 0.1 µl
For example 0.1 µl has 2 cells (total no of cells/4)
1 ml or 1000 µl has (2x1000)/0.1= 2x104 cells/ml
Viable cells/ml= Average viable cells/ square x dilution factor x 104
Dilution factor= total volume/volume of sample