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POLLINATION & FERTILIZATION LAB
Overview of Lab:
Experiment 1 is the most time consuming part of the lab. It requires 5 time points
that are spaced 30 minutes apart. Start this experiment right away and do the
other experiments between the time points.
Goals:
1. Manipulate pollen tube growth in vitro.
2. Dissect flowers to identify critical reproductive organs and cells.
3. Learn how to do manual crossing of self-fertilizing plants.
Experiment 1. Determine the optimal boric acid concentration for tobacco
PT germination and growth.
Pollen tubes are heterotrophic. They need to import nutrients and energy from
their surroundings to support their astounding growth rates. In all species
examined, pollen tube growth rates are much faster in the pistil than they are in
vitro. For many years, researchers have tried to develop pollen tube growth
media that will lead to pollen tube growth rates similar to those observed in the
flower.
Boron is known to be essential for pollen tube growth in vitro and is required for
proper cell wall formation.
Today, you’ll determine the optimal concentration of Boron (Boric acid) for pollen
tube germination and growth of tobacco pollen tubes.
You will be provided with petri dishes containing pollen tube growth medium
supplemented with the following:
1. 0 boric acid
2. 0.15 mM boric acid
3. 1.5 mM boric acid
4. 15 mM boric acid
For each of the four treatments:
1. START THIS AS SOON AS POSSIBLE! Inoculate the medium with tobacco
pollen by touching an anther to the surface of the medium. You can probably
use a single anther for multiple Petri dishes. Use the dissecting scope to make
sure that at least 20 pollen grains have been deposited. Record the time of
inoculation for each treatment.
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2. After 30 minutes record the % germination and the length of the longest
pollen tube. You can do length measurements using the digital camera
and NIH image. If you are using the dissecting scope equipped with a digital
camera you will want to use the highest magnification (6.3 x). For the inverted
scopes 20x works very well.
3. Continue making measurements every 30 minutes until the end of lab. Our
goal is to inoculate at ~2:15 and take 5 measurements (2:45, 3:15, 3:45,
4:15, and 4:45)
Data to collect as a group for each [boric acid]:
% Germination (examine at least 20 grains per time point)
Tube length (either longest tube or average length of 10 or more tubes)
Questions:
1. What is the optimal concentration of Boron in pollen tube growth media?
2. What is the role of Boron in pollen tube growth? Is it required for pollen tube
germination, tube elongation, or both?
3. The formula weight of boric acid (H3BO3) is 61.83. How would you make 100
mls of a 1 M stock solution? How much of this would you use to make 100 mls of
media for each of today’s experiments?
4. Were the concentrations of boric acid used in our experiments sufficient to
determine the optimum? Which concentrations would you try next to increase the
resolution?
Experiment 2. Flower dissections and manual crosses.
We will have several tobacco plants in the lab. You are free to dissect and cross
them as you please. Manual crossing is a fantastic way to understand plant
reproduction.
These plants are self-fertile; so, you have to remove the pollen before it is mature
otherwise the plant will self-fertilize before you’ve had the chance to add pollen
from a different plant.
What you should do:
1. Dissect young and old flowers and fruits.
2. Examine these under the dissecting scope. Find and Draw:
 Anthers
 Pollen
 The stigma
 The style
 The ovary
 Ovules
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 Seeds
3. Determine which stage of development produces mature pollen grains.
4. Emasculate at least one immature bud and pollinate it with pollen from a
different plant. As a negative control, leave one emasculated bud unpollinated.
As a positive control, mark a bud that you will allow to self-fertilize. Mark your
cross using tape (ask for help) so you can follow the progress of your cross over
the next few weeks.
MATERIALS NEEDED FOR EXPERIMENTS:
Plant Material: Tobacco plants
Reagents:
Basic Pollen Tube Growth Medium:
[Final]
[stock solution]
18% sucrose
crystals
1 mM CaCl2
1M
1 mM Ca(NO3)2
100mM
1 mM MgSO4
500mM
0.5% Noble Agar (Difco) powder
0, 0.15 mM, 1.5 mM,
or 15 mM Boric Acid
4% (0.65 M)
Other gear:
Microscope slides 25x75x1mm
Cover slips 22x22mm
Stage micrometers for calibrating length measurements.
10 cm ruler
H2O
DAPI staining solution (0.1 M sodium phosphate, pH 7, 1 mM EDTA, 0.1% Triton
X-100, 0.4 µg mL–1 DAPI)
p1000, p200, and p20 pipetman and pipet tips
sharpies for labeling
tape for labeling
forceps
dissecting microscopes
One dissecting microscope with camera (works to measure tube length)
Compound fluorescence microscope
two inverted scopes (best for measuring tube length)
two upright scopes
digital camera (Qimage)
Q capture software
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Scion image analysis software
How to Capture an image:
USING Q capture:
1.Open Q capture software
2. Acquire>live preview
Basic format = monochrome; preview depth = 8 bit; capture depth = 8 bit
Make sure that you capture all of your images at the same magnification.
Make sure you note the pixel resolution. I suggest using 1360x1036 pixels
3. Acquire>snap – this will create a tiff of the image in your live preview;
there is also a camera icon on the toolbar that snaps.
To make the whole image fit in one window: view>display settings>zoom>
50
4. Save pictures to my documents/bio44/lab 3 data/your group name
USING Olympus software:
1. click on Olypus DP Controller icon
2. click on the ‘capture’ tab
3. click on the blue arrow to start live window
4. the metal slider near the biuonocular eyepieces on the scope needs to
be out to send light to the camera
5. click on ‘auto exposure’
6. focus and adjust exposure time (need to switch to ‘manual exposure’ if
you want to change exposure time).
7. Click on the camera icon to snap your image
8. Save as to my documents/bio44/lab 3 data/your group name
Measure length using Image J:
 Choose the freehand line tool from the tool bar (right click on the line tool).
 Use the line tool to mark the length of the pollen tube
 Analyze>measure (cntrl M)
How to determine the scale of your images (by calculating it):
Focus in on the stage micrometer and snap a picture using the same
magnification and pixel resolution you used to take pictures of your pollen tubes.
The stage micrometer has markings on it that are 100 m or 10 m apart. The
total length of the marked region is 2.2 mm. Open the image in image J and use
the line tool to determine the length in pixels of a defined length on the
micrometer. Convert pixels to m and use this conversion to convert your tube
lengths from pixels (the output of image J) to microns. I will post an example of
how this is done in an excel spreadsheet.
Using ‘Set Scale’ in image J so it will measure length in real m.
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Image J collects length data in pixels (or image inches in the case of the
Olympus camera). We want to measure pollen tube length in microns (mm).
You can train image J to give you lengths in microns by following these simple
steps.
1. Set up the microscope exactly as you will use if to take pictures of pollen
tubes.
2. Make sure all of the image settings (dimensions in pixels, bit depth) are
the same as those used to take pictures of pollen tubes.
3. Focus in on the stage micrometer and snap an image
4. Open the image using image J
5. Use the line tool to draw a line from the outside edge of one of the bars on
the stage micrometer to the outside edge of another bar. Note the ‘actual
distance’ you have measured.
10 m
100 m
Line to measure 200 m
6. Click analyze>set scale
7. A dialog box appears:
8. The ‘distance in pixels’ should be pre-filled – this
should be the length of the line you drew
9. Type in the know distance – in this case its 200
mm
10. Tell it the units should be microns
11. Hit OK
12. Now, image J will give you lengths in microns.