a small grain that consists of a few cells. To the naked eye it appears as
a yellowish (pale yellow) dust-like substance that is either dispersed by wind
formed within the sacs (or microsporangia) in the anthers that are
located in plant flowers. The development of plant anthers also involves the
growth and differentiation of tissue that develop to produce the pollen sacs.
This is then followed by a process of cell division (meiosis) that results in
the formation of clusters referred to as quartets within the pollen sac
The quartet then undergoes further divisions to produce pollen, which
serves as the male gametophyte of plants (seed plants) for reproduction.
Depending on the type of plant, pollen is either dispersed by wind or insect to
the receptive stigma.
* A plant
that is pollinated through wind is referred to as anemophilous while those by insects are known as entimophilous.
* Once a pollen grain falls on the
receptive stigma, it starts absorbing water and becomes activated. This causes
the gametophyte in the grain to develop a tube reaching the ovule of the
flower, which in turn allows male gamete cells to be transported to the ovule
viewing pollen grains under stereo microscope, it is advisable to view treated
pollen (washed using a little alcohol) and untreated grains separately
in order to see the difference.
The procedure involves the following simple
Wash the pollen grains
using alcohol and place the sample on a microscope slide (also keep an unwashed
Add 50 percent glycerine to
the sample and place under the stereo microscope for viewing
may also place a healthy plant anther with pollen (without treating it with
alcohol or adding glycerine) on the stage of the stereo microscope and try
observing how it appears
grains have an oily surface that can be easily removed using alcohol.
Therefore, alcohol may be used to remove the oily layer and thus get a better
look at the grain surface. Fifty percent glycerine is used for
the purposes of hydrating the grains and making them swell for good viewing.
viewed under the stereo microscope, pollen grains will appear as grossly
shaped, irregular structures/particles. However, the shape and appearance of
the grains will vary depending on the type of pollen under investigation. For
untreated grains, there is poor contrast compared to treated pollen grains.
The glycerol jelly technique is one of the most common mounting techniques used for
pollen. It involves using glycerol jelly, which consists of 10 grams of
gelatin, 35ml of distilled water as well as 30 ml of glycerol.
possible results, students are advised to use freshly collected pollen.
Freshly collected pollen
A compound microscope
IPA solution (Isopropyl
Microscope cover slips
Place a small amount of the
sample (pollen) into a small dish and add a few drops of IPA solution for about
10 minutes (isopropyl alcohol will remove the oily layer from the surface of
the grains. If the pollen had not been removed from the anthers, the solution
also helps isolate them from the panther to only retain the pollen grains)
Using warm water, melt the
glycerine and allow it to remain warm
Obtain 1 or 2 drops of IPA
and pollen mixture using a dropper and place at the center of a clean glass
slide (prepare two slides)
Place the slide on a
hotplate for about 2 minutes to dry
Add one drop of the
glycerine jelly on to a microscope cover slip and lower the cover slip gently
at an angle on to one of the prepared slides to remove air bubbles and bring
the jelly into contact with the pollen
Allow the slide (with
jelly) to stand for about 5 minutes on a hotplate - This allows the glycerine
to penetrate the pollen grains.
Place the slide in a cool
area until the jelly sets (for this technique, a small amount of nail polish can
be added to seal the sides allowing the preparation to be stored and used for
View the two slides under
viewed under the microscope, the stained slide is clearer because of better
contrast. The grains will appear as tiny ovoid particles with what seems like a
scaly surface or ornamentations.
The unstained slide appears more transparent /translucent
and does not give a clear view of the grain surface. However, the appearance of
the grains is also largely dependent on the type of plant from which the pollen
Wet Mount Technique
Add two drops of glycerol
on to a clean slide
Add a small sample of the
pollen (this can also be done by tapping the anther to obtain the pollen)
Gently place the cover slip
on the to sample at an angle to remove air bubbles
Nail polish can be used on
the sides for the purposes of sealing
Place the slide on the
microscope for viewing
* For this
technique, toluidine blue or acetocarmine may be used for staining the sample
Dry Mounting Technique
the simplest method that involves the following simple steps:
Place the sample on to the
microscope glass slide
Cover the pollen sample
with a cover slip
View under the microscope
Viewing Pollen Tubes under Microscope
tubes are important structures that grow down the style allowing for gametes to
the transported to the ovary for fertilization. To view pollen tubes under the microscope,
Farmer's solution and sodium hydroxide can be used.
Mimulus ringens flowers (with
Alcohol, Farmer’s solution
0.4M sodium sulphite
1 molar sodium hydroxide
8 M NaOH, or a 10 M NaOH
Aniline blue, aniline blue
with fluorescent brightener or acetocarmine combined with acidified aniline
blue (either stain can be used to stain the sample)
electron microscopy, acetolysis is one of the most popular methods used for
preparing pollen. However, it has been shown to cause distortions, which
influenced the development of a new and better method. One of the best and most
recent methods involves the use of Aerosol-OT and amyl acetate.
involves the use of acetic acid for the purposes of breaking down compounds
Carefully remove the
anthers from the flower and immerse them into 3 percent Aerosol-OT solution for
Carefully remove the pollen
from the anthers and place them into a 15 ml centrifuge tube
Keep the pollen sample in
Aerosol-OT solution for about 5 days for rehydration
Using the pipette, drain
out the Aerosol-OT solution from the tube and add water for about 10 minutes
After 10 minutes, drain out
the water and add the acetone/water mixture (1:1) for about an hour
Lightly centrifuge the tube
to concentrate the pollen grains
Place the tube in an
ultrasonic bath for a minute to sonicate the grains
Drain out the mixture
solution and replace with a fresh mixture and allow to stand for about 1 hour
(This cleans out any extra material on the surface of the pollen grains)
Replace the acetone/water mixture
with distilled water and allow to stand for 10 minutes
Remove the distilled water
and add 50 percent ethanol and allow to stand for an hour
Replace 50 percent ethanol
with 70 percent ethanol for an hour
For periods of 1 hour,
replace the 70 percent ethanol with 90 percent ethanol and 100 percent ethanol
(repeat 100 percent ethanol twice) for dehydration (for the second round with
100 percent ethanol, allow to stand for about 4 hours)
Drain out the ethanol and
add amyl acetate for about an hour (this is done to prepare for critical drying
Remove the pollen grains
from the tube and transfer them to a modified BEEM capsule
Cover the ends of the
cylinder and press the cylinder in to the cap
Carefully remove amyl
acetate so as to retain pollen
Using a pipette, transfer
the pollen grains into the basket
Place the baskets in the
critical point drier
Remove the upper lid and
invert the opened cylinder to mount the grains on studs
To make the grains float
down the surface of the stud, one may gently tap the basket
Before viewing, the grains
should be shadowed with gold
viewed under the SEM, the grains can clearly be seen as inflated or deflated
structures (of varying shapes) with rough surfaces or cleavages depending on
the type of pollen being viewed.
When viewed under the microscope (compound
microscopes or electron microscopes) the grains may have different types of
ornamentations with the ornamentations being irregularly distributed on the
surface of the grains.
For instance, whereas some of these ornamentation may be
visible all across the surface of the grain, others may only be present on
polar ends of the grain or across given sections on the surface of the grain.
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