Before we could extract the pollen, we first had to gather the pollen, which all team members gathered in the wilds from many different trees and plants.
At first, we gave the male syncarps of birch, willow and spruce in a plastic tube, which was electrostatically loaded on its walls. During vortexing, the pollen stuck to the walls and the syncarps were collected at the bottom of the container. Afterwards, the pollen was wiped from the walls with cotton buds and the cotton pellets with the pollen were vortexed. Finally, we checked the pollen using light microscopy if they were separated correctly according to their plant species.
Opening Pollen
In order to isolate and to be able to analyze the DNA, we tried to open the pollen in different ways. We used ribolyser tubes and small ball mills used to homogenize biological samples. We also added trypsin to pollen in order to decompose parts of the exine (outer layer of pollen). Another idea was to use liquid nitrogen to cool down the cell walls and to break them open.
To form a trypsin solution, we resuspended 20µg of trypsin in 200µl of H2O. Then we split up the solution into 4 eppis with 50µl each, having approximately 5µg of trypsin within every eppi. According to our proportionality-calculations, we then added pollen to the trypsin and incubated at 37°C overnight. The following day we analysed our samples with light microscopy, enlarging by 100 and 400.
To use ribolyser tubes we added 10, 20 and 50 mg of pollen combined with 400 µl of water into the tubes. In the next step, we shook the tubes in the ribolyser and centrifugated them at 110 rpm for 10 minutes. The results were two different layers, one brighter than the other. White shroud was on the top layer. After abstracting the light-brown, lower layer with a chemical dropper, 400µl lyse buffer was added and the solution was centrifugated for 1 minute at 11000 rpm. Afterwards, we used the machery nagel plant Nucleo spin III Kit for the DNA extraction.
Finally, we tried to open the pollen with liquid nitrogen. Therefore, we put pollen in a small bowl and added liquid nitrogen. Then we cracked the pollen using mortar and pestle and checked the results of all approaches with the light microscopy.
pollen of a spruce opened with nitrogen with 400x magnification (light microscope)
Electron Microscopy
There are two different kinds of microscopes:
Light Microscope
Electron Microscope
While the light microscope is a tool used in basic biology lessons in school to see plant cells and seeds, the power of a light microscope was not enough to check whether the pollen was cracked or not because the pollen cores are too small. To see better results, we needed an electron microscope.
An electron microscope uses a beam of electrons to create an image of objects. Electrons are thrown on an object and are reflected towards a detector. The given data are analyzed to form a picture.
That would have helped us to see the success of our experiments- if it had worked so far.
Gallery
Here is the result from trying to open the pollen with trypsin and a ribolyser.
pollen of a willow with 400x magnification (light microscope)
pollen of a spruce with 400x magnification (light microscope)
pollen of a birch with 100x magnification (light microscope)
