Sunflower sectioning....

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mrsonchus
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Re: Sunflower sectioning....

#31 Post by mrsonchus » Wed Nov 01, 2017 11:56 pm

Hi all, nothing much to report tonight, having sectioned and mounted another dozen or so slides I still haven't come across the next clearly delineated stage of development that I'm after - the 'heart-shaped' stage. Some more globular-stage embryos but that's about it. Still, it's all good sectioning and staining practice, and I've still many more sections to cut before I exhaust this batch of wax-blocks...

So a few more images not much different from the others, although I haven't found any more embryos with the 'flat-topped' (see previous post to this one) beginnings of the heart-shaped stage either.... :cry:

Here's another globular-stage embryo sitting in the bottom of the ovule,
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and,
ws_tvqs-0025.jpg
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Still at least I've managed to back-off the Fuchsine that was so heavily over-staining these embryos - I used good-old Safranin instead!

Here are a few more of those 'twin-cell' hairs found on the epidermis of the immature pericarp,
ws_tvqs-0009_001.jpg
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This bright blue is 'Alcian-blue' stain - a good stain for cytoplasm as a contrast to Safranin (and Fuchsine as it happens).

Sorry there's not much to tell, I'll have some more sections cut tomorrow - I'll keep burrowing into those wax-blocks until I find the next stage/s - or ultimately make some new ones of more advanced Sunflower embryos - if I can see any in my specimen jars - I do still have a lot of Sunflower tissue in fixative so the chances are pretty high....

Back soon. :D :)
John B

MichaelG.
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Re: Sunflower sectioning....

#32 Post by MichaelG. » Thu Nov 02, 2017 12:30 am

Work that good is never tedious to watch, John.
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MichaelG.
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mrsonchus
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Re: Sunflower sectioning....

#33 Post by mrsonchus » Fri Nov 03, 2017 1:19 am

Thanks Michael, appreciated.

Well, I've been mounting some of Wednesday's sections and haven't found the heart-shaped embryo stage - but, I have been lucky enough to have several slides showing the very, very earliest stage of the embryo. The fertilised egg-cell becomes a zygote, which then begins it's transformation to a fully-formed embryonic plant within a seed - awaiting germination.
The very first cell divisions of the zygote form the 'suspensor' - the 'stalk' essentially atop of which the embryo develops. We've seen the 'globular' stage and the very start of the 'heart-shaped' stage, but I now have a few images of the stage immediately after formation of the suspensor and before formation of the 'sphere of cells' that is the globular stage.
This stage of very first cell-divisions of the embryo proper consists of only 2 or 4 cells!

Here's a picture of this extremely early stage - this is before the previously seen globular and early heart-shaped stages... This is stained with Safranin-O and Alcian-blue counterstain - I love the bright Alcian-blue - but if not applied and removed very quickly (say 5 seconds max) if will render the Safranin dull.
Image

Here's a nice image of the cells of the ovule - the nuclei can be seen suspended by the cytoplasmic skeleton - rendered this stringy-form by the fixative, in particular the acetic-acid component. This is often called the 'acid fix figure' I believe. I like it for the detail and the stain colours which are pretty bright yet clear.
This tissue is stained with the marvellously metachromatic 'Toluidine Blue' - a sort of (aqueous) 'all in one' that stains in different colours - usually pinks, reds, purples, light blue and darker blue shades - also an excellent stain for live tissue. For permanently-mounted tissue it has to be handled carefully - if it encounters a slightly alkaline solution (such as with tap water) rather than de-ionised water during the rinsing and dehydration stages it simply 'blues' and become monochromatic. Here it has performed very nicely and maintained its differentiation right through to mounting.
Image

Here's a nice Safranin and Fast-green staining of the ovary's funiculus that connects it to the parent plant and runs into the side of the integument that folds over the ovule.
Image

Here's a whole achene and ovary stained with Methylene-blue - another metachromatic stain related to Toluidine-blue - it too is very sensitive to pH and easily loses it's metachromasia if rinsed with a trace of alkali. If again added strong and fast the effect is far better than a long and weaker exposure (staining time).
Image
and,
Image

I thought I may as well post these extra images as I have used a few different stains that some may not have seen very often. The trick is to stain to maximise contrast and brightness whilst avoiding the loss of detail that often occurs. Sometimes it's good, sometimes not - but I've improved my staining lately and am pretty please with 'where I'm at' - it's a very long learning curve - but such fun and so rewarding. :D :D

Still plenty more sections to be cut from that set of tissue-blocks - I'll have some more made soon - hopefully with some interesting morphology to show you - at the very least I'll have a practice with some more stains I think!

Back soon. :D :D :)
John B

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zzffnn
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Re: Sunflower sectioning....

#34 Post by zzffnn » Fri Nov 03, 2017 1:39 am

Beautiful work, John B. You did it again!

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mrsonchus
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Re: Sunflower sectioning....

#35 Post by mrsonchus » Fri Nov 03, 2017 2:21 am

Thanks zz' having a great time with these tiny embryos! :D

A clarification of what I think I rather 'messed-up' a little in the earlier post/s - the distinction between the 'funiculus' and the 'suspensor'....

The funiculus is the 'seed-stalk' that attaches the seed/achene/cypsela to the placenta of the parent plant .
The suspensor is the tiny first-stage of cells in a 'stalk' that joins the embryo to the nutrient-supplying endosperm - effectively the food supply of the developing embryo. The elongating (by cell-division) suspensor pushes the embryo up into the endosperm fully.

Here's a labelled image - apologies for the earlier somewhat ambiguous text - I'm learning as I go!
This should make it a little clearer...
ws_labelled-whole-embryo-1.jpg
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Hope that makes it a bit clearer - it's helped me too! :oops:
John B

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billben74
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Re: Sunflower sectioning....

#36 Post by billben74 » Fri Nov 03, 2017 10:33 pm

Always a joy. And always amazing and facsinating inights in both histology but biology too.
Love the early embryo without polarity you have discovered.
Many moons ago I wrote few essays in my finals about development. Mainly animals but really interesting to see the process here "in the flesh".
Now I'm wondering if plants use a homobox genes to define initial polarity like fruit flies and us... may need to go and google.

Wonderful wonderful...

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