Sunflower sectioning....

[mrsonchus]

Hi all, I've been looking at some Sunflower tissue lately and over the past few days have processed some developing (immature) Sunflower ovaries and have 13 wax-blocks ready to section - as well as quite a few other sections from Sunflower tissue of recent study.
I haven't posted much material for a while so I though I'd better put a little into this fine forum.

This late (too late actually) Summer I planted and grew several nice 'giant yellow variety' Sunflowers, with a sly eye on their innards for the lab
Here is a nice fresh set of Sunflower ovary wax-blocks ready for the attention of the 'Mighty Shandon' - hopefully tomorrow if I get a chance!
First - this is the protocol used - it needed to be a 'full-on' three-day protocol as the seeds are quite hard and somewhat difficult to penetrate with reagents and especially wax during the final infiltration phase in the wax-oven.

ws_sunflower seed protocol.jpg (72.69 KiB) Viewed 4673 times

The 'crowded' block at the bottom is what I call a 'storage block' for unused (at this time) fully-processed tissue (seeds in this case) that is simply re-embedded at a later date if I need some more sections of this tissue.
I sliced off the top of some of the seeds to facilitate penetration, safe enough as the 'action end' is the end by which the seed is attached to the flower-head - this is the end from which the embryo begins to develop. Others I partly sliced longitudinally pre-processing for the same reason. The rest and majority I processed whole, but tried to make sure a long protocol ensures proper & complete processing - if it's incomplete the sections will, together with the blocks whence they came, be scrap!

ws_sunflower seed oct 2017 (2).jpg (67.26 KiB) Viewed 4673 times

I've already made some slides from the Sunflowers' leaf sections, petioles, stem/leaf nodes and roots that I will put up a few images of hopefully later tonight - oh yes - I've some nice slides also of the flower-heads with their multitude of immature florets - before they are developed into florets let alone the reproductive structures and ultimately seeds - it's interesting to see the primordial flower structures emerging from the actively dividing floral meristems - images to come of these also, they're the first sections I've taken of floral apical meristems and their associated primordia....

Back soon hopefully.

[JimT]

John, welcome back and looking forward to all your image posts. Except the first one of the white board steps - made my head hurt

You are really serious about this and it shows in your work..

JimT.

[mrsonchus]

John, welcome back and looking forward to all your image posts. Except the first one of the white board steps - made my head hurt

You are really serious about this and it shows in your work..

JimT.

Hi Jim, and thank you my friend for the encouragement.
Having a rummage for some quick images of the other sections to add a little colour and interest 'til I section these seeds - one batch is at room-temp right now, the other at 4-deg C - tomorrow should be good for some early sections.

Ah - the first step - I tend to abbreviate and write like a drunken spider sometimes....
It just means the first post-fixative step (the tissue can remain in fixative, AKA 'FAA', virtually indefinitely and so I don't count it as part of any protocol) is a rinse in alcohol (OH) of the same strength as that in the fixative - in the case of my favourite FAA formulation that's 50% - in order to rinse away the other ingredients (in the case of FAA they are formalin and acetic acid) prior to beginning a dehydration series through several stages of increasingly concentrated OH (in de-ionised water up to 'pure' 95%) as a precursor to the next stage moving into 'Histoclear', the clearing-agent (which is a wax ante-medium also)...

Thanks for your interest my friend.

[billbillt]

Hi John B.,

YES!!!... I am glad to follow along with this new adventure.. Your projects are always interesting and thought engaging... Can't wait!...

BillT

[mrsonchus]

Here are a couple of those wax-blocks with the fully processed and wax-infiltrated (right down to the intra-cellular & molecular level for the wax-matrix to support the tissue adequately for clean and thin microtomy) with wax, ready for the microtome!

They look promising - thus far - fingers firmly crossed as always at this stage.....

ws_sunflower seed oct 2017 (5).jpg (26.43 KiB) Viewed 4656 times

The left-hand seed has had it's tip removed as mentioned above, the dark ends are the tops of the soon-to-be seeds with mature embryo within, and at this pre-embryo stage this tip is safe to remove - I hope!

Another block,

ws_sunflower seed oct 2017 (7).jpg (24.42 KiB) Viewed 4656 times

The middle seed is darker and more mature than it's neighbours - the florets and consequently seeds mature 'from the outer to the inner of the large disk that is the glorious head of a Sunflower - consisting actually of up to hundreds of these tiny 'florets' as per the Compositae in general....

smaller-ws_sunflower-immatu.jpg (156.51 KiB) Viewed 4656 times

Here's an image of an early and quite thickly-cut 'morphological' slide of such an immature flower-head in LS before florets have developed - the meristems ('growing tips') that produce the 'stem cells' if you like, for each individual floret are visible together with the floret-organ primordia. These develop 'from the outside in' in terms of the flower-structure also, as in outer calyx (usually the bud's protective leaf-jacket) primordia first, then the corrolla (petals), then the reproductive structures such as the stamen-filaments, ovary and pistil - and these are seen sequentially appearing as primordial buds 'left behind' by the meristem (producer of the new undifferentiated primordia-cells) - fascinating to be able to see this pre-floret stage followed by the ovary then seed stages!

I really must choose a larger flower than the Compositae have - trouble is I love Compositae! Some may remember my early adventures with the dreaded Sonchus!

Anyway a couple of images from those rough sections,
Right across the immature flower-head - apologies for the small and very thin image..
These are not seeds - they are the beginnings of the flowers, long before seeds are built!

Closer-in to the primordia and meristem,

smaller-labelled-6micron-su.jpg (270.39 KiB) Viewed 4656 times

Last of the 5 - the outer leaves protecting the entire flower-head is, as is the entire Sunflower plant, extremely hairy! What amazes me is the sheer variety of trichome types that the Sunflower has - quite easy to see in this image I hope,

smaller-sunflower-immat-flo.jpg (264.96 KiB) Viewed 4656 times

If you're wondering about the colours - they are a few of the many different staining protocols that I generally practice with or try-out with such early and rough sections as these - plus it's just great fun! please don't adjust your white-balance - these sections are meant to be these colours. Once I get a tissue-block sectioning how I want it to I usually settle down to the very reliable and indeed attractive Safranin (maybe Fuschine) and Fast-green classic-combo that never seems to fail!

Back tomorrow hopefully with some sections of those seed tissue-blocks. Hope you like these to be going on with...

John B.

[mrsonchus]

Hmm, just thought, you may like a look at the immature flower-heads as I prepared them for processing using the larger (double volume of the white cassettes I most often use) red tissue cassettes. You can see just how small the sunflower heads are at this primitive stage,

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ws_DSCN9014.jpg (149.33 KiB) Viewed 4651 times

No wonder the florets are still virtually primordial!

Here are a leaf-blade and petiole image to make up the five, rough but OK.
Under the knife....

ws_DSCN9019.jpg (63.8 KiB) Viewed 4651 times

Rather poor leaf-blade section, I've yet to make some more I'm afraid....

ws-sunflower-leaf-ts.jpg (112.5 KiB) Viewed 4651 times

A nicer leaf-stem (petiole) section though,

ws-sunflower-leaf-petiole-T.jpg (185.67 KiB) Viewed 4651 times

Back tomorrow.

[apochronaut]

What a terrific pile of beautiful, precision, work, John. I especially like your lunch boxes!

[mrsonchus]

Hi apo' - thanks, your a generous fellow! Pleased you like them, they're fascinating to make - more to follow from the Sunflowers soon.


Lunch-boxes! Yes, they're just like teeny bananas aren't they!

[mrsonchus]

Hi John B.,

YES!!!... I am glad to follow along with this new adventure.. Your projects are always interesting and thought engaging... Can't wait!...

BillT

Aha, Bill my friend - nice to hear from you! Thanks Bill, having a lot of fun and fascination with these Sunflowers! More soon..
John B.

[JimT]

You are the Master, fantastic. Really like the explanations for each step. Waiting for the next "Lesson".

[Crater Eddie]

Great new adventure!
CE

[vasselle]

Bonjour
Félicitation pour votre travail
Et les images de sections sont très belles.
Merci pour le partage
Cordialement seb

[billben74]

Lovely to see your work again.

Sadly don't have the time at the moment to use slicer, but very much enjoy vicariously.

[mrsonchus]

Thanks for your kind comments folks, here are a few early sections, just had time last night to take a first-cut from one block containing a single cypsela.
You may think of these as seeds, cypsela is just a term for a dry-fruit (which is what these actually are, as in all compositae), specifically 'cypsela' also implies an inferior-ovary (that is, developing below the petals etc - like a Daffodil's ovary or a Fuchsia).

Anyway, late last night I took a first section or six at 10µ which just passed (in LS) through the 'shell' as it were, not (yet) penetrating into the interior containing the ovule/embryo.. A good indicator for the processing of the tissue - as this 'casing' is the hardest part of the cypsela, and the most likely to be ruined if the processing or sectioning is poor. Luckily this one sectioned very nicely without any signs of processing problems, fully infiltrated and supported by the wax matrix.

This is the cypsela in the block as sectioned, after 10-12µ 'roughing' and fine 1-2µ 'polishing',

ws_DSCN9330.jpg (105.65 KiB) Viewed 4606 times

After I cut the essentially paradermal first section/s, I dried, stained and mounted one quickly, stained with Safranin and Fast-green and containing three of these paradermal LS sections.... A very promising result thus far - fingers crossed - again!
Here's a complete-section stitch to give you an idea of 'what we're dealing with'....

ws_sunflower-cypsela-first-.jpg (219.16 KiB) Viewed 4606 times

I particularly like the clean sectioning right along the lengths of the trichomes - a good sign for sections to come I think (hope),

ws_sunflower-cypsela-tricho.jpg (165.56 KiB) Viewed 4606 times

and closer-in,

ws_stacked-trichome.jpg (47.74 KiB) Viewed 4606 times

I'm pretty pleased thus far, but we've yet to pass through this outer 'shell' into the nitty-gritty of the developing embryo (hopefully) within each cypsela, including of course this one....

Today I had a chance to cut some more sections and pass through the casing into the 'innards' and the embryonic structures that I'm after for good slides... Back later with some raw section-in-wax images as I hadn't the time this evening to similarly dry, stain and mount the new sections. But, I do have some images where the inner embryo's tissues seem to be emerging, taken at three thicknesses, reducing as I felt I was getting to the optimal sectioning-plane through the tissue. Basically from 10µ to 8µ down to 5µ as I 'arrived at the best sectioning-plane' as best as I could judge it to be at this early stage.
Back soon in next post with some images, stay with the adventure chaps, it's looking promising - I've about 50 cypselas for sectioning in 13 tissue-blocks - somewhere in that lot are bound to be some lovely orientations and images.....

Back later. John B.

[mrsonchus]

Aha! A few 'in the wax' images - we're entering the interior of the cypsela's casing now - let's see what we find - sorry these are all in the wax and frankly still wet! Should get time tomorrow to stain and mount a few slides....

Anyway, here's the sectioning-sheet,

ws section details.jpg (122.43 KiB) Viewed 4605 times

All I had time to cut today I'm afraid,

ws_first sections.jpg (185.15 KiB) Viewed 4605 times

Closer-in we see a little structure through the wax,

ws_DSCN9325.jpg (90.03 KiB) Viewed 4605 times

and,

ws_DSCN9326.jpg (82.74 KiB) Viewed 4605 times

ws_DSCN9328.jpg (84.65 KiB) Viewed 4605 times

Looks OK - looking forward to mounting these, and sectioning some of the many others to come!

What fascinating things plants are!

[billbillt]

Hi John B.,

Off to a wonderful and interesting start !..

BillT

[mrsonchus]

Hi Bill, as they say - "The game's afoot" my friend.
I've been able to stain and mount 15 slides from that first block, which is unfortunately a little poor in terms of orientation. Not that this matters as these early sections have served their primary purpose very well. That is to say I've been able to see that they section nicely, the processing has been thorough etc.
I do have a few that have a little info within, but although the inner embryo is seen, it's form is obscured by this poor orientation.

So, I started with the knowledge that the sections are structurally sound and 'ready to stain and mount' - the next step therefore being to make some trial staining runs to see exactly which protocol may prove to be the best for these sections and the many to follow.... As the next step I work through the de-waxing process - that is the removal of the wax from the now sectioned and dried tissue affixed firmly to their slides, using 'Histoclear', a non-toxic Xylene replacement that I have used from day 1 of my slide making exploits.

As each section is cleared of wax and then washed with OH I stain them briefly with a quick dollop of Safranin in OH just to allow me to take a quick look at the structure/s contained in each slides tissue - those that are sub-standard or have no useful views within may then simply be thrown in the bin at this stage, and a lot of time saved staining and mounting those of no use is saved.

So, I have ended-up with 15 slides stained and mounted from this first single-tissue block - only 1 or two are of any permanent value as mentioned earlier, but I've been able to perfect a very nice staining protocol for the many sections to follow these, with a view to showing the intricate details that I expect (hope...) to be visible when better oriented sections start to be taken.

So the method to here is basically,

1) Sections in wax, on slides, dried and firmly attached. These are seen in images in my earlier posts.
2) Slides bearing sections are placed into 'Histoclear' wax-solvent until the wax has completely left the sections, usually about 20 minutes is just right.
3) These Histoclear-soaked slides are then similarly washed and immersed in two changes of about 5 minutes each of pure (well, 95% anyway) alcohol (Isopropanol) to remove the Histoclear.

Now we have our de-waxed sections in OH (alcohol) ready to take either a path through re-hydration via a multi-stage water (DI) alcohol series to pure DI, ready for aqueous staining, or straight into staining with an alcohol-based stain....

Here are some images from some of the very first sections, grossly over-stained with Safranin and fast-green to begin with.... At this very heavy level of staining only the main morphological features are seen properly - finer details of nuclei, chromosomes etc are totally (well almost) obliterated by the excessive staining. This is however a useful stage as it gives a good indication of how to proceed with the finer points of staining and differentiation of finer tissue details with these particular sections.

ws_sunflower early embryo (2).jpg (129.33 KiB) Viewed 4551 times

and

ws_sunflower early embryo (1).jpg (77.31 KiB) Viewed 4551 times

These are starting to reveal the developing embryo - poorly orientated and over-stained, but still of value for assessment,
Closer-in the spherical 'ball of cells' look as though it may be the 'head' if you like of a developing embry - unable to be sure though. Very little detailis visible as the stain is making just about any fine details - nothing that can't be fixed with a few staining and differentiation alterations...

ws_sunflower early embryo (5).jpg (57.79 KiB) Viewed 4551 times

and the other section on the same slide of the same tissue,

ws_sunflower early embryo (6).jpg (59.03 KiB) Viewed 4551 times

Even with a little post-processing there's little I can do for these....

ws_embryonic-cells-collage-.jpg (115.64 KiB) Viewed 4551 times

That's the 5, back soon - supper is calling me! Stay tuned!

[mrsonchus]

I hope to be able to post a few images later, and tomorrow probably start-in on sectioning the multi-tissue blocks that I have,
the ones ready at 4-deg C (in the refrigerator in fact)....

ws_DSCN9341.jpg (43.08 KiB) Viewed 4548 times

and this lot at room-temp,

ws_DSCN9342.jpg (66.7 KiB) Viewed 4548 times

These are the slides made from that first single-tissue block, hopefully I can post some more images of my alterations to the staining-protocol, as I now have it just right for maximum detail! Hopefully later tonight...

ws_DSCN9340.jpg (48.42 KiB) Viewed 4548 times

[Francisco]

Magnificent. Thanks for sharing

[billbillt]

I always love your series of plant slides.. Your written narrative is the best and keeps the interest flowing!... Will follow this and all of your series with great interest..

BillT

[mrsonchus]

Well, I've had a look over those slides and haven't really found what I'd call a good view - so here are a few images of about the best-bits in this set - hoping for better with the rest of them - should definitely be some good morphological images of the embryo-entire when I section some of those.

All I really have are these few images, mainly of cell-division of the embryo cells - sorry they're a little unremarkable at the moment...

Here as is clear, I've drastically reduced the staining of Safranin and as a results more detail is visible. It's tempting to interpret this structure as the embryo's 'axis' - but I really don't feel confident that this is it. I suspect this apparently axial shape is just an artefact of some oblique orientation - still, in other respects things are coming along nicely - but to me this is just 'a long-looking shape' at this time.... The mass of surrounding tissue is the Sunflower ovule's single integument. although some vascular differentiation may be visible in the red-stained strand at top right...

ws_28_10_17_286.jpg (90.35 KiB) Viewed 4533 times

Here are some mitotic cells that are (I hope) reasonable to look at. These are cells of the very early pro-embryo dividing as far as I know....

ws_dividing cells.jpg (56.44 KiB) Viewed 4533 times

and, dividing cells of the embryo's surrounding endosperm,

ws_anaphase embryo mitosis.jpg (75.66 KiB) Viewed 4533 times

ws_interphase embryo mitosis.jpg (43.95 KiB) Viewed 4533 times

Well, not much to enjoy really with this one-tissue block, but my hopes are high for the rest of the batch! I'll try to make some time to begin sectioning those tomorrow - back soon!

[billbillt]

Thanks John for posting these interesting slides of this stage of mitosis.. I see them as very good...

BillT

[Radazz]

WOW! Really nice work, John.

[mrsonchus]

Thanks Bill & Radazz, pleased you like them. I can't wait to get into the other blocks - I'm pretty certain there will be some good orientation - out of all those tissues!
Hopefully I'll get some sectioning-time tomorrow... Thanks both for your interest and encouragement.

[mrsonchus]

Good news! Having searched through my books and the internet I've realised that I have failed to realise just how immature this embryo is - which puts a completely different interpretation onto the images and their correctness of form as they are shown - for the better I'm pleased to say

There really isn't much too wrong with the orientation - the achene was lying pretty 'flat' in the bottom of the mould - i.e. has been sectioned in a nice longitudinal plane as suggested by the very first section that is shown early in this thread as paradermal - as it hadn't actually penetrated the hard 'shell' - actually the dried fruit or 'pericarp' (a shrivelled and dried-out ovary-wall in fact) of the Sunflower.

What these sections show is not a poorly-oriented mature embryo complete with two fat cotyledons (fat in the Sunflower as the food reserves are all in the these seed-leaves - no endosperm reserves are left in the mature embryo of the Sunflower's seed) but a pretty well oriented but very immature embryo - which doesn't even possess cotyledons yet!

What my sections actually show are the very early developmental stages of the embryo - consisting primarily of the 'stalk' that connects the developing embryo to it's life-support system - the parent plant's capitulum (the big wide fleshy disc upon which the Sunflowers florets, then seeds, sit), a pair of integuments that wrap over and protect the developing ovary - these later dry to become the very thin, papery in the Sunflower, true seed-coat, and the recently formed tiny embryo....

I've been examining these slides expecting to see a pair of cotyledons, a root-tip and apical shoot, when all the while I failed to realise that what I have is the developmental stages way before that stage - and should be looking therefore for integuments, a funiculus (the life-support 'stalk') and a tiny immature embryo with no recognisable differentiated parts of the mature embryo yet developed!
That realised, these images and slides now make perfect sense, but it'll take me a while to put all that new information into this thread....

A picture will help,
back soon - I'll need to label an image or two with what I've just learned above...

[mrsonchus]

Hi all, well after the somewhat confusing and limited results from the first block, I have been able to take some more sections from several of the others, most are actually pretty good, with some nice very-immature embryo stages just discernible.
Having realised after a lot of reading the other night, that these embryos are nowhere near maturity and the stages at which features such as cotyledons will be developed and visible, I have stained and mounted a few today which I think you may find interesting - especially as the embryo's very early 'spherical stage' has been captured - one I certainly have never seen before - these structures are truly tiny.

Here's a collage image of a couple of full cypselas - remember the stripey 'Sunflower-seed' with which we're all familiar is an fact a fruit - as are all Compositae 'seeds', yes including my old favourite the Sonchus!
This fruit (formed from the ovary's pericarp) isn't a fleshy part like say an Apple, rather a dry seed-like 'case' or 'cypsela'. When a Sunflower 'seed' with the stripey exterior is opened the little grey-ish soft edible part is the actual seed - the often quite papery but always extremely thin covering of this seed is the 'seed-coat' proper.

Sunflower cypselas immature fruits in LS, with seeds containing very early embryo....

ws_embryo-collage-1.jpg (490.53 KiB) Viewed 4428 times

I suspect that the cypsela on the right may be an unfertilised egg-sac as the large ovoid structure is right against the bottom of the lining - once fertilised a 'stalk' (the suspensor) develops and atop this will be the very early 'pherical stage' seen in the left-hand image. This spherical stage has yet to develop polarity - a 'top' and 'bottom' - eventually the root and shoot - and the next noticeable stage will be the development of the two cotyledons, beginning with two projections that will give the embryo the appropriately-named 'heart-stage' as it looks, with it's two cotyledon buds, like a heart-shape. Hopefully a few embryos of the remaining tissue-blocks will exhibit this stage for us!
The florets (and therefore embryos) of the Sunflower mature 'from the outside in' on the flower-head and immature cypselas taken (as are these) will vary from totally unfertilised and undeveloped to later and further developed stages as the peripheral tissue is examined.

These sections may look a little more purple and blue than my usual Safranin + Fast-green versions, that is because for speed I used the very fast-acting basic-Fuchsine (the red/purple) with my newly arrived and mixed Alcian-blue, a beautiful alternative to Fast-green and an excellent counter-stain to the reds of Safranin or as here Fuchsine.

ws_Untitled_Panorama1.jpg (320.16 KiB) Viewed 4428 times

Closer-in to the spherical embryo - apologies for the over-staining,

ws_x25-tcam-5mp-1msec.jpg (238.18 KiB) Viewed 4428 times

and,

ws_embryo-spherical-stage-1.jpg (207.98 KiB) Viewed 4428 times

Finally, a picture of a 'failed block' that you may find of interest. These tissues were as the protocol posted at the start of this thread shows, 'given the full treatment' in terms of the length of processing and the frequency of reagent-changes throughout the entire histological sequence - taking about 3 days not including the fixative stage, to complete.
The reason I sliced the tops off of some of the darker and harder cypselas pre-processing was, as mentioned in an earlier post, an attempt to try to improve the chances of full processing and wax-infiltration of these trapped-air prone little 'boxes' that are the cypselas.... I also included a long early vacuum-stage for the same reason.
Now, nearly every section has cut beautifully, but there is one block in particular that clearly had not processed fully due to trapped air within the seed's 'locule' and as a result has not sectioned properly when the seed's interior was breached by the sectioning process.

The white-area/s when closely examined under my dissecting 'scope is indeed an air-containing void and the block will not section, but is still of interest.

ws_incomplete-infiltration.jpg (197.06 KiB) Viewed 4425 times

Still, I really do have many more blocks ready to section, so no worries about this one! As I section the other blocks I'll keep you posted with further progress reports and plenty more images as and when (fingers crossed...) more interesting structure is revealed - back soon!

[billbillt]

Mr. John B.,

You have really pushed it over the top.. I have never seen such wonderful detail of an embryonic stage... You have a gift of creation in your endeavors with the "Mighty Shandon"!.. I am following with the intense interest...

BillT

[MicroBob]

Hi John,

thank you for showing your work in this detail! It is very interesting to see which steps you take and what the results are.

Bob

[MichaelG.]

Great work, John

Lovely sections and helpful description of the work.

MichaelG.

[mrsonchus]

Thanks fellows all - I'm very encouraged by your kind comments.
Some good news! After perusing the dozens of new sections for likely candidates I've found one or two that indeed have the next stage on from the spherical or 'globular' stage - not vastly different but definitely a progression of the embryo's development.

Now, here are a few images of the one precious slide I have, with two sections on it of an embryo in the start of the very next stage after the one pictured already as the 'globular stage'. The next developmental stage is the 'heart-shaped stage' where the embryo - as it's name suggests - begins to resemble a heart-shape in LS, as seen in my sections here.

Just as this tiny and very immature embryo - still about the size of a pinhead, begins to differentiate, the cells from the spherical 'globular' stage begin to 'flatten' at the top as a precursor to entering the full-on heart-shaped stage. This very brief and subtle 'inter-stage' if you like, with the flat-top embryo, is what I have in the following images, next to the globular-stage for comparison and with a few parts labelled.

There were only two sections really showing this shape out of about 200 taken and about 30 more slides part-made to the 'in wax on the slide' stage - where I gave them all a good examination and ultimately came up with one single slide bearing two sections! Still, worth it I think, and I've many more tissue-blocks of these Sunflower cypsela/embryo tissues to section yet. I'm hoping my next set of sections will 'turn-up' perhaps a fully-formed heart-shaped embryo - that would be a real treat as I love following this minute process (of embryo development) along with the making of permanent slides of their fascinating structure.

As you can see, one section is 'purple-ish' and the other is 'red-ish' - I varied the amount of acidified Harris-Haematoxylin used on each section - easy enough to do on the one slide with careful use of a couple of fine pipettes and a steady hand.
Unfortunately the dratted (and now I will say infamous!) HH contained quite a lot of bits of Haematin pigment 'lumps' as it often does, requiring it to be filtered before each use - a step I rather stupidly forgot to take... Still, although this one precious slide has lots of mucky pigment-chunks all over it - none have (amazingly!) gone onto the very parts of the tissue in which I'm interested - namely the embryo and related structures. A bit of luck!
I really don't like the heaviness of this stain for this dense tissue - I should have known better and used Safranin I think - still, without mistakes we don't learn much - I hope....

Here are the images, see what you think,
this one is in it's embryo-sac and labelled in case you may like to know the names of some of the parts, please forgive any mistakes, I'm learning as I go...

ws_labelled-embryo-in-sac.jpg (334.35 KiB) Viewed 4374 times

Now, the 'flatness' of the embryo about to 'sprout' cotyledon-tips at it's upper corners and become the 'heart-shaped stage' I'm after proper, is pretty nicely defined considering.

But, this one doesn't show what this next (other of the only two thus far) one does, the 'stalk' that connects the developing embryo to the Mother-plant during it's development - rather like the umbilical-cord of a mammal you might say.... This 'stalk' is called the 'suspensor' and can be seen in this red-ish section, and is of course labelled. I hope these labels are correct, they're the best I can do with my very limited knowledge, so please let me know if you see an error.

Here's the suspensor-bearing section next to the earlier-seen still spherical 'globular' stage from yesterday's post for comparison. These are entirely comparable views as both are in the same sectional-plane, that of a longitudinal-section through the embryo-sac and embryo...

ws-edited embryo-stage-comparison-.jpg (359.75 KiB) Viewed 4327 times

All I really have time for tonight. Hopefully I'll have time tomorrow to cut some more sections in pursuit of the truly 'heart-shaped stage' - back soon when I have some more of interest. Hope you're not finding this rather lengthy thread a little tedious - there's just so much for me to drone on about!
Back soon!