Student Sheet 4

www.saps.org.uk

Pollen Tube Growth

Technical and teaching notes
Safety
Care should always be taken when handling and mixing chemicals.
Wear eye protection and consult CLEAPSS HAZCARDS for full information about different
chemicals.

Preparation of materials
Recipe for Brassica Pollen Germination Medium
Mineral Salt Solution to make 1 litre:

0.417g calcium nitrate (Ca(NO3)2);

0.2g boric acid (H3BO3);

0.101g Potassium nitrate (KNO3);

0.217g magnesium sulphate (MgSO4.7H2O);

3.5 cm3 of 1.0M ammonium hydroxide (NH4OH).

Sugar solution

1.2M sucrose (41g sugar in 100 cm3 of distilled water)

Equal volumes of these two solutions should have a pH of 8.8. Vary the NH 4OH in the first
solution to adjust the pH.

Note
Using this method with pollen from other species, Butler published a list of the best flowers
and the best concentrations to use throughout the spring and summer. You will find this in
Butler K.G. "Pollen germination across the seasons" School Science Review 2000 82 (298)
p93f. You can read a copy of this article on the SAPS website.

Background Information

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When a pollen grain is released from an anther, it is partially dehydrated. If it lands on the
surface of a stigma of the same flower (self-compatible) or of a different flower of the same
species (self-incompatible), it rehydrates. Some species have dry stigmas, and it is thought
that lipids in the surface coat of the pollen grain bring about a concentration gradient of water
that directs the growth of the pollen tube. If the humidity is very high, the pollen will rehydrate
anyway. Other plants have wet stigmas and the stigma exudate hydrates the pollen grain
itself, so surface lipids in the pollen are not so important.
The growth of pollen tubes is easily observed under medium power of a microscope (see
below), but the in vivo growth is often much faster because of several factors that are known
to enhance the growth. Some of these are calcium, boron, and sucrose: this last compound
acts as a source of carbon as well as exerting an osmotic effect. Some species produce
flavinols in both the pollen and anther, which is necessary for growth. Mutant maize plants
that are deficient in the enzyme that synthesises flavinols are self sterile. Addition of flavinols
restores the ability of the pollen to grow and fertilize the ovules of the female plant.
As pollen tubes grow, callose plugs are produced close to the aperture of the pollen grain, so
the cytoplasm becomes concentrated towards the growing tip. Pollen tube pictures are
available on the SAPS website.
Pollen allergens
Many people suffer from hay fever and other respiratory illnesses brought on by inhaling
pollen. The cause of this is certain proteins present in the pollen grain. The pollen specific
genes for these are very similar in a wide range of plants, and their base sequences are
highly conserved, suggesting they play an important role in pollen development. The proteins
coded by these genes are show similarities to pectate lyases, and it is thought that they
enable the pollen to partially dissolve tissue in the style of the female flower, so the pollen
tube can successfully travel down to the ovary. Other allergens found include profilin (in birch
pollen), which binds actin and is therefore important in developing the architecture of the
pollen tube (cytoskeleton and microfilaments). Grass pollen contains expansin like proteins.
These are involved in cell wall expansion. The presence of proteins in pollen can be seen
under the microscope by staining with Coomassie Blue or similar. Put a drop of water
containing pollen on a microscope slide and place a cover slip on top. Place a few drops of
stain, containing some sucrose or glycerol, at one side of the cover slip (these compounds
have an osmotic effect on the pollen grain causing the cytoplasm to ooze through the
apertures. Place some tissue paper at the opposite side and allow the stain to percolate
through the pollen. The grains within the emerging cytoplasm of the pollen will be stained
blue. These are the proteins.
Observing pollen tube growth
This has a reputation of being unreliable, being dependent on the age of the pollen, the
species being used, and the constituents of the growth medium. The SAPS protocol given
here can be unsuccessful with some flowers. A simplified method, using just sucrose (0.6 mol
dm-3), works very well with a number of plants commonly found in school grounds. These are
in flower in the different seasons, so the experiment can be undertaken at various times in the
school year.
The table below indicates plants that have proved successful.

Month

Successful

March/April

Birch

Not successful

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April

April/May

Cotoneaster

Fraxinus

Crab Apple

Cherry

Ceanothus
Viburnum

May/June/July Chestnut
Mountain Ash
July/August

Lime
Indian Bean

Chestnut pollen will produce rudimentary pollen tubes even when incubated in water alone,
so one suspects the actual concentration of sucrose used is not critical, though it may
influence the speed of growth. We have produced images of the growth over a period of one
hour: These are available in the zipped file available on the SAPS website so students can
analyse the data to work out the rate of growth of the pollen tubes. This lends itself to
investigative work at GCSE and post-16, with variables such as sucrose concentration,
temperature, pH, and the addition of mineral ions.