Pollen and pollen tubes
Pollen
Introduction:
Pollen is the male gamete of seed plants (Angiosperms and Gymnosperms). Because it is nonmotile, plants must rely on outside agents to transport their pollen from the male part of one flower
(or cone) to the female part of another.
This transfer is called pollination. Commonly, wind or animals (mostly insects, but also birds and
mammals) transfer pollen and the characteristics of the pollen grain reflect the pollination method.
Pollen storage
Fresh is always best but sometimes it is necessary to collect pollen when it is seasonally available
for later use:
 collect mature anthers that are shedding pollen naturally. The pollen should be naturally
dehydrated
 either place whole anthers or the collected pollen in small air-tight vials and store in a
freezer for later use
Some plants that produce copious amounts of pollen may be useful here, eg. Lilies, sweet corn
(Zea mays) and many cone-bearing gymnosperms.
Mounting pollen
Very little preparation is required (simply dust the pollen onto the mounting medium and add a
cover slip). If the pollen is large clumps it will need to be separated by stirring. A small needle is
ideal for this. Stirring also helps to get rid of any air bubbles that are attracted to the pollen.
However, the mounting medium may affect the pollen and storage potential of the slides:

Air mount i.e. no mounting medium, simply dust onto a slide and add a cover slip.
This is ideal for wind pollinated plants if the pollen is fully dry.
It may be difficult to focus under high power however.
Slides can be sealed with nail varnish along the edge of the cover slip and be stored for
long periods

Water is an ideal medium for temporary slides.
However dry wind pollinated pollen soon rehydrates and the ‘foldable’ nature of the pollen
grains will be lost.
Some animal pollinate pollen is sticky or oily and in these cases it is difficult to remove all
the air bubbles when mounting pollen in water.
Water mounts should not be sealed and kept.

Glycerol or 50/50 glycerol/water mixture.
These fluids can be used for animal pollinated pollen. Glycerol on its own causes some
dehydration if the slide is kept for long.
These media are also useful if the slide is observed for a long time as the heat from the
microscope lamp will not dry the slide out as quickly.

Glycerol and alcohol mixtures. This works similarly to the above medium but with two
advantages: the oils present on many pollens is dissolved away and alcohol act as an
antiseptic so this mixture should be used if slides are to be sealed with nail varnish and
stored.
Images (next page):
Daffodil: Medium Power (x100, left) and High Power (x400, right)
Camellia: High Power (above and below x400). Note the drops of oil on the pollen grain surfaces
Rhododendron: Low Power (x40, left) and High Power (x400, right)
Gymnosperm: Low Power (x40, left) and High Power (x400, right)
Foldable structures
The images of the Gymnosperm pollen above show a common characteristic of windblown pollen.
Because windblown pollen is exposed to dry environments, it tends to dehydrate. This also means
it will be lighter and more easily transported by the wind. The pollen is in a dormant state during
pollination but once it reaches the stigma it quickly re-hydrates and resumes activity i.e. growth of
the pollen tube towards the ovule and the egg cell.
As the pollen dehydrates, it shrinks and the structure of the pollen grain wall allows it to fold in on
itself without damage i.e. the pollen grain wall collapses in a controlled manner.
In the image above some pollen grains have not dehydrated as much as others and have not yet
fully folded in and collapsed.
Lily Pollen:
The two photographs on this page illustrate the added detail that can be gained by focusing up and
down as you observe the specimen especially under High Power.
Above: a surface focus view shows the characteristic surface markings that many types of pollen
have. Below a equatorial focus view reveals tiny droplets of fluid that help the pollen stick onto its
pollinating insect. (both photographs at High Power x400)
Pollen tubes
Whichever method of pollination is employed by the plant, the objective is to transport the pollen
grains from the anthers to the stigma (of another plant). The stigma surface is covered with a
concentrated solution of sugars (mostly sucrose). This has two functions:
 It makes the stigma sticky so that pollen grains will adhere
 Sugars have a high-energy content. The pollen will need to use this on the next part of its
journey.
Once the pollen grain rehydrates it begins to produce a pollen tube. This tube contains a ‘sperm
nucleus’ and is carried by the developing tube down the style to the ovary. Inside here, the sperm
nucleus will fertilise the waiting egg cell. The resulting zygote will grow into the embryo plant inside
the seed. The ovary wall itself will become the fruit.
Growing pollen tubes in the laboratory.
Pollen tubes can sometimes be induced to grow even though they are not on the stigma. It is a
matter of providing them with the appropriate environmental conditions. These include:
 Sucrose of the correct concentration, start with a stock solution of 20% sucrose and dilute
as required.
 Other factors (often lipids) that ensure that only pollen grains of the correct species grow
towards the ovule.
 Nutrients required by the pollen tube. These include Boron (B) and Calcium (Ca).
The recommended concentrations are:
B
100 mg/L Boric acid
Ca
300 mg/L Calcium nitrate.
One recipe suggests a stock solution of 10 g honey, a small crystal of boric acid and a
small smear of marmite made up to 100 ml.
Many plants will produce pollen tubes given sucrose only. The other requirements are not always
vital so it is worth trying your luck.
It also pays to use pollen from flowers at different stages of development; pollen that is not
mature may not respond and grow pollen tubes. Usually mature flowers give best results
There are two recommended methods.
1.
Using a spotting tile:
This method is easier but there is the
possibility of damaging the pollen tubes as
they are transferred to the microscope slide
afterwards.
In each well provide a sucrose solution with a
different concentration by diluting your (20%)
stock solution. Suggested concentrations
are:
20% (4 drops stock)
15% (3 drops stock + 1 drop water)
10% (2 drops stock + 2 drops water)
5% (2 drops stock + 6 drops water)
2% (2 drops of 5% + 3 drops water)
Sprinkle pollen into each solution and stir into the solution.
Use a small sheet of glass to cover the whole tile to prevent desiccation and keep warm (200C)
Leave for 1-6 hours, transfer a drop of each solution onto a slide, cover and observe.
2.
Using a Petrie dish as a humidity
chamber
In this method, the pollen tubes grow on the
slide itself.
Prepare 5 petrie dishes as shown. There is
thin film of water in the bottom of the dish so
that your slides will not desiccate. The lid is
used to maintain humidity.
The slides are held up above the water by
short lengths of drinking straw (squeeze
them slightly to stop them rolling).
Each slide has a drop of sucrose solution
Dilute your stock solution as in the spotting
tile method.
Sprinkled each solution with pollen and mix the pollen in with a needle.
Add a cover slip and keep warm for 1-6 hours.
Remove the slide carefully and observe.
Images:
The following four photographs show germinating Camellia pollen tubes. Iodine stain was used to
enhance their features.
Pollen tubes just emerging from the pollen grains. High Power (x400)
A mass of pollen grains and tubes. Medium Power (x100).
Note the random directions of pollen tube growth.
Individual Camellia pollen tubes. High Power (x400) above and below
Positive (+ve) chemotropism in pollen tubes
It has often been noted that when pollen tubes are grown in this way they grow in random
directions. Yet when on the stigma they must direct their growth towards the ovary. It is possible
that some positive chemotropic response is responsible for this directional growth.
The hypothesis can be tested by introducing a small, dissected fragment of the ovary (or perhaps
of the style) under the cover slip as the pollen tubes are being grown (using the petrie dish,
humidity chamber method). It will take 1 – 6 hours to grow, depending on temperature.
Small piece of ovary tissue
Do the pollen tubes still grow in random directions of do they now follow a chemical trail (given off
by the ovary or style tissue) that leads them towards the ovule?
Pollen tube nuclei
Pollen tubes have 2 types of nuclei that may be visible:
 Pollen tube nuclei which are responsible for the growth of the pollen tube itself
 Sperm nuclei, which are the ones that are involved in fertilization once, the tube reaches its
destination inside the ovule.
To see these nuclei it is necessary to stain the pollen tubes with a stain such as methylene blue or
aceto-orcein.
Above: Lily pollen tube stained with methylene blue shows a tube nuclei (arrowed).
Below: Lily pollen tube stained with aceto-orcein stain showing a sperm nucleus.
(both photos: High Power x400).