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Doug,
I am not an expert in fertilization biology in plants but I
am not going to let that stop me from conveying conventional wisdom.
Plant ovules probably do exert some sort of chemical signal but as
far as I am aware no one has ever been able to use it to manipulate
pollen tube growth direction in culture. I believe the ideas in the
field now are that the extracellular matrix (what we plant types call
the cell wall) provides essential contex for the chemotaxis to work.
What people do now is a kind of sort of dissection where they take
the whole female flower, open it partly so the tubes are growing down
the style and the ovules are splayed out a bit. But I think these are
deeply tricky experiments wih a low success rate. If your client has
been taking part in this kind of research, then fine. But if he or
she thinks he or she can set this up as a quick and dirty assay, hmm,
I wouldn't bet on it.
One more point. Agar is mostly water. So when you put a small
object on agar it is immediately enveloped in a film of water. You
can image samples on agar without a coverslip, but of course as the
mag goes up, the optical quality goes down.
My few pollen grains,
Tobias
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>I could use some advice with a sample preparation question for
>confocal that was recently posed to me by a plant scientist. He
>would like to look at the fertilization of egg containing ovules
>using GFP tagged pollen tubes in arabidopsis. The dissected
>portions of the appropriate plant parts (I'm not a botanist) would
>be placed on an agar surface; pollen tubes actively grow on the agar
>surface and enter the ovule to fertilize the eggs .
>
>The question becomes how to best image this process on our upright
>Zeiss LSM510NLO-Meta. My colleague would like to avoid the use of a
>culture liquid, since the liquid may diffuse whatever chemotactic
>agent draws the sperm into the ovule.
>
>I'm thinking that a dry lens (somewhere between 20x & 40x), with
>decent working distance might be in order. A coverslip could be
>placed over the plant parts and agar (to prevent them drying out
>over the 8 hr time period). What I can't figure out is what to do
>with the probable air gap under the coverslip if we can't fill the
>space with an aqueous solution. Glycerine?
>
>Note, an inverted scope would mean imaging through the agar. While
>my colleague did this earlier with a wide-field microscope
>time-lapse of the GFP labeled pollen tube, I'm wary of doing this
>with a confocal.
>
>Any ideas?
>
>Thanks,
>Doug
>
>--
>^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
>Douglas W. Cromey, M.S. - Assistant Scientific Investigator
>Dept. of Cell Biology & Anatomy, University of Arizona
>1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA
>
>office: AHSC 4212 email: [log in to unmask]
>voice: 520-626-2824 fax: 520-626-2097
>
>http://swehsc.pharmacy.arizona.edu/exppath/
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--
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