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Notes to the Teacher:
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Class time needed:
One double period or one single period will
be needed, depending on time available and teacher preference.
If double periods are available, the students
may germinate the pollen themselves. If double periods are not available,
the teacher should start the germination process before class. It
may take from 1 to 3 hours to germinate the pollen, depending upon the
temperature, maturity of the pollen, and other unknown factors. We
suggest using pollen from several different flowers to insure obtaining
pollen that will germinate during the desired time period.
If you have a double period, prepare
the pollen growth chambers and start the germination process at the beginning
of class While pollen is germinating, review the parts and functions
of the flower, germination, pollination, etc. Check the pollen after
about 45 minutes. If pollen tubes are forming, proceed with the lab
activity.
Teacher Preparation Time:
1. Prepare agar plates and growth medium - 20 minutes (Students
may prepare the plates,
depending on the class and the time constraints.)
2. Prepare growth chambers if you will not have your students
complete this step - 20 minutes
3. Start the germination process if you will not have your students
complete this step - 20
minutes
4. Shopping for or collecting lilies
Student Grouping:
Students may work in pairs for this exercise.
Helpful Hints:
1. If you have limited time, you may prepare the slides and plates
ahead of time and fix them
with either of the following fixatives:
Fixative #1 - Acetic Alcohol -- glacial acetic acid
: ethanol (1:3)
Fixative #2 - 10% ethanol
Drop one of the above fixatives on the slide or plate.
2. We recommend using lilies as a pollen source because they are available
year-round and they
provide copious amounts of pollen that is easily
removed from the flower.
3. Realize that not all of the pollen will be at the proper developmental
stage for immediate
germination. We have found that using
at least three different flowers will assure
germination of enough pollen grains for observation
during the lab.
4. For your information, calcium and boron have been shown to
enhance pollen tube formation.
The tube grows from the tip and a calcium-ATPase
may be responsible for providing the
energy for this rapid growth.
5. We have written this lab protocol to investigate the importance
of nutrients (calcium
and boron) on pollen tube growth. If
you wish to use this lab solely to view pollen tubes and
cytoplasmic streaming, we recommend germinating
the pollen on Medium B or Medium D.
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NOTE: Media C and E are used for extensions, only.
Medium A - 10% sucrose
Medium B - 10% sucrose, 100mg/L boric acid, 300 mg/L calcium
nitrate
Medium C - 10% sucrose, 100mg/L boric acid, 300 mg/L calcium
nitrate, 200 mg/L
magnesium sulfate, 100 mg/L potassium nitrate
Medium D - the same as Medium B with the
addition of 1% agar. Heat to boiling and pour into
petri dishes.
Medium E - the same as Medium C with the addition of 1% agar.
Heat to boiling and pour into
petri dishes.
Preparation of Growth Chambers:
Growth chamber consists of a petri dish in which is placed a bent glass
tube or Pasteur pipette. Water is placed in the bottom of the dish
and a slide is placed on top of the bent glass. When the dish is
covered, this maintains a humid environment to promote growth of the pollen.
The teacher may prepare these growth chambers, or the students could bend
the glass and prepare the chambers themselves.
Other Materials and Equipment:
Microscopes - compound (dissecting microscopes may be used also, if
available)
Microscope slides
Lilies - at LEAST three different flowers
Fixatives - see recipes
This activity helps students answer:
1. How the sperm arrive at the ovum in plants
2. Requirements for pollen tube growth
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Purpose
To investigate the germination of pollen tubes in vitro and
to investigate the importance of calcium and boron in the growth medium.
Equipment
Growth chambers
Microscope slides
Agar plates
Growth media
Lilies with mature pollen
Compound microscope
Procedure
1. Preparation of growth chamber:
Obtain a petri dish and a glass tube. Carefully heat the middle
of the tube in a Bunsen burner and bend the tube into a "V" shape.
When the glass is cool, place it into the dish and add a thin layer of
water to the bottom of the dish. Place a microscope slide across
the bent tube and add the cover.
2. Germination of pollen in liquid medium:
Carefully place a drop of liquid medium A and B onto opposite ends
of the slide in the growth chamber. Carefully remove an anther containing
mature pollen from a flower and gently touch it to the surface of medium
A first and then medium B. You should see the pollen float onto the
surface of the drops. Cover the chamber and set aside. Check
after about 45 minutes for signs of germination.
3. Germination of pollen on agar:
Obtain a petri dish containing medium D. Carefully remove an
anther containing mature pollen and gently drag it across the surface of
the agar. You should see pollen adhering to the agar. Check
the plate for germination after about 45 minutes.
4. Make observations:
Observing petri dish - the petri dish can be opened and place on the
stage of the compound microscope to view the pollen grains. Be careful
not to get the objective lens into the agar!
Observing the slide from the growth chamber - remove the slide from
the growth chamber, dry the bottom of it, and place it on the stage of
the compound microscope. Do not add a cover slip, but be very careful
not to get the objective lens wet.
Observation
1. Draw what you see at three different times, being certain
to record the length of the pollen tube at each time, as directed by your
teacher. Remember to include the magnification and the diameter of
the field of view on your drawings.
2. Look for and describe cytoplasmic streaming. Record
when you see this event. (If the slides have been prepared ahead of time
and fixed, you will not see this.)
Conclusion
Write a conclusion to this lab that includes the following information:
1. Compare and contrast the pollen tubes from the different preparations.
2. What do you think accounts for the differences you see?
3. What did you see that you did not expect to see?
4. What did you expect to see that you did not see?
5. What differences would you expect between pollen germination
in vitro and in vivo?
6. What further experiments could you design to find out more
about pollen tubes?
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Brewbaker, J.L. and Kwack, B.H. 1963. The essential role
of calcium ions in pollen germination
and pollen tube growth. Am. J. Bot.
50:859-865.
Messerli, M. and Robinson, K.R. 1997. Tip localized Ca2+
pulses are coincident with peak
pulsatile growth rates in pollen tubes
of Lilium longiflorum. J. Cell Sci. 110:1269-1278.
Shivinna, K.R. and Rangaswamy, N.S. 1992. Pollen Biology. Springer-Verlag, New York, N.Y.
Steer, M.W. and Steer, J.M. 1989. Pollen tube tip growth. New Phytol. 111:323-358.
Wang, C., Rathmore, K.S., and Robinson, K.R. 1989. The responses
of pollen to applied electrical
fields. Dev. Biol.
136:405-410.
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