Results and discussion for WEC - 2011 RUN # 3 (ROSA GIGANTEA)

Technique Embryo Culture
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21

Well…they have transformed into this, after a 12 hour water immersion:



These seem to have germination potential…almost there…!!

I just sowed them back into pre-wetted perlite, only I got real tired of handling the foam cups like cotton wool, and went back to a glass cup (one gazillion times easier to seal off with cling wrap, very minimal adhesive tape and without disruption of the embryo+perlite contents).

I also have a feeling that for the purposes of WEC, glass may actually create a more effective fogging/misting/condensation in the interior of the sealed container, maybe this has something to do with the different physical properties of glass compared to foam (an insulator).

Anyways, here is the WEC set up now:



I hope these embryos are happy to grow into seedlings, now!!!

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~36 hours later, here is how they look now (in bright sunlight, they are actually greener than these pictures suggest…kind of yellow-lime green, especially near the cotyledon/radicle fusion “V” point, but not all parts of them are this color, some parts are still white, which worries me!!!):





And here is one of them when I immersed it in water just a few minutes ago in order to see if there still was this bubbling I was talking about further up (= gas being actively produced by the embryo I assume, but I cannot be 100% certain of this). This bubbling caused this one to swim on the surface of the glass of water:



The other embryo had less bubbling, and mainly sunk to the bottom of the glass of water, but occasionally it too floated to the top as well. I wonder if this bubbling is a new sign I can actively watch out for, when trying to assess the health of these embryos as they progress through these water treatments???

At this point in time, these are viable and have potential to progress to seedlings. Although visible chlorosynthesis is now showing, both are actually progressing rather slowly!! These two are definitely fairly “dormant”, (but nowhere near as dormant as “waxy” embryos).

I worry that these two embryos were still not receiving optimal humidity at the surface of the perlite, where they were sown (despite pre-wetting the perlite, leaving a slight water level at the base of the cup, and sealing the cling wrap well with adhesive tape).

Sooooo…I decided to actually mist the inside-facing side of the cling wrap before I re-sealed it. This step might hopefully encourage more humidity to be distributed to the surface zone of the mix, where the embryos are sown.

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George, this is how I germinated my ‘Test Tube Baby Love’ except instead of using perlite I used potting mix. I added some wet (soggy) potting mix to a microwave safe bowl, covered it and nuked it on high for 10min. I added small amounts of this sterilised soil to small sterile medicine cups and let them cool, covered in cling wrap. When cool the embryo, which had been soaking to help remove the testa, was placed radicle down in the potting mix, sprayed with Mancozeb, and the cling wrap was again placed over the wet mix and I left it. The embryo was placed on the glass over one of my aquariums for light from 3 x 6500K fluoro tubes and warmth. Took about 3-4 days to open and green up and and a few days more for the first leaf to start to appear. Each day I removed the cling wrap for under a minute to respray with Mancozeb. I forget whether I diluted it or not; sorry… it was nearly a year ago now. When the first green leaf appeared I potted it into a 3" pot in more sterile mix and kept it under lights and covered to harden it off then after a week or so it was put out in the greenhouse. Its filled a 10" pot now and lives outside with all the other roses.

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George

Just a suggestion

I think you would be able to manipulate the moisture levels in the perlite simply by raising or lowering the water level in the glass container. Since both the water level and the embryo would be visible through the glass, you could raise it untill you get the desired moisture level at the embryo. If it goes too high simply draw some off with a hypodermic needle inserted below the surface to make adjustments,(a few trials would sort it out). That way you could have complete control of the situation, (I think)

I take a real interest in the trials you do George but since I have never even managed to get one embryo to look like germinating, let alone grow. Feel free to ignore what I say.

Russ

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Hi Russ!

I always try to carefully read and understand people’s opinions/ideas here.

Please feel free to add any ideas, they are all welcome, be they from you, or from anyone who reads here, RHA member or otherwise!!.

Yes, I have tried what you suggest. Oddly, what seems to happen is that the water still concentrates to the bottom as the perlite floats, and the layer of perlite above it seems to act as a layer that blocks efficient rising of water vapor from the layer of water it is floating on. No amount of extra water on the bottom has seemd to make a great difference to the ability to get droplets forming on the cling wrap (which is what I was aiming for).

I am finding that by misting the inside surface of the cling wrap, water droplets are immediately formed on the inside of the cling wrap and these are maintained on the inside surface of the wrap, and water is therefore being trapped on the surface zone by the same phenomenon it seems to be trapped in the bottom layer (ie. the perlite mass seems to be able to create 3 chambers in the glass if enough water has been added to it, to casue it to float some):

  1. The bottom layer is water, 2. The middle layer is wet perlite, and 3. The top layer is air plus water droplets/vapor one adds with the spray mister.

I am very pleased to say that these glass cups are now maintaining an excellent droplet number on the cling wraps due to this extra step of spraying the inside surface of the cling wrap before it is sealed off!!!

…lets see what happens.

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Hi Simon!!

I started embryo cultures using JEC (jar embryo culture) several years ago now, which was also a relatively “sterile” method, (like perlite might be considered).

As a matter of fact, I had been using seedling mixes for the initial germination phase of WEC for over one year now… It had been great when it worked, and then when something went wrong with it, depending on the batch used, it turned into a universal catastrophe. I need much more consistency / control in matters with WEC, than seedling mixes have provided in my own experience. It is just too bad.

Actually, Don Holeman’s suggestion of utilizing pure perlite so far seems to have the potential to be optimal in relation to WEC. Thanks Don!!!

I have no idea about other methods of embryo culture, where one uses antifunfals, peroxide etc, and the interaction with seedling/potting mixes…I imagine there would be more complex interactions going on there, and cannot comment in any meaningful way. There is no direct correlation with WEC and other methods of embryo culture where such chemicals are being added.

27

These two were never going to make it easy for me.

Here they are 24 hrs later:



I am seeing green growth emerging from their apical meristem region (rudimentary true leaves)…so I am calling these “germinated”… yet there are no signs of a radicle growing out (ie. top growth has not matched bottom growth)!!!

Yes, another case of the dreaded “radicle blunting”, most likely going on here!!!

:frowning:

LOL…

I have developed my own “remedy” for this…sometimes it has worked for me, the alternative is to watch them get green leaves and no roots, and die in the perlite!!!

I plant these radicle blunters in seedling mix, making sure their cotyledons are int he air/light, and their radicle tip is totally buried in the dark mix. I have no idea why this works somethimes!!

Off to the fresh batch of seedling mix and to pot them up right now!

28

Here are two more achenes:



Here are their extracted seeds:



Here two more again:



…and again their extracted seeds:



Embryo shots will follow soon.

29

Here are the embryos from the above 4 seeds:



If you look carefully, you can just see how the one which is the fattest of the lot (top most) has suffered a slicing injury to one cotyledon, however it is a type of incomplete “flap injury”, and all its embryo parts are still intact, including this flap segment!!! LOL!!!

Unfortunately, none of these four actually appear to be 100% pearly whyte types, when viewed under bright light, however none were waxy/extreme dormant types, either.

I am predicting a very low germination rate, purely on these color characteristics.

Time will tell.

For now they are in a three day water soak (water will be changed daily).

30

LOL.

I wish I knew how to add arrows and circles to emphasise difficult-to-see things (like this flap I referred to in the above example), in photos!

If anyone want t teach me, my email is:

gvarden AT bigpond DOT NET DOT au

31

Here are the last 4 R.gigantea achenes I kept:



…and here are the extracted rose seeds, each one placed below its respective achene:



The one to the extreme right of screen already looks to me to be dead, however I will give it a try in the three day water soak, as it cannot be ignored at this rather late and desparate stage of the game!!!

To be quite honest, extracting these R.gigantea rose seeds is easier than you would think.

Oddly, the main problem I have found with this batch of R.gigantea is not to do with the embryo extractions at all. It is to do with the fact that the embryos nearly all are acting as though they have sustained some sort of severe “damage”.

They are not waxy/dormant embryos as they are mainly white types. But usually white type embryos easily pop out of their testa coverings like there is some lubricant that helps them to “slip out” real easily, once a large enough opening is made for them to slip out. Unfortunately nearly all of these R.gigantea embryos are not showing this fast and easy readiness to “slip out”. Instead, they just sit in the water with their papery coverings half removed, and the majority do not slip out, even after several hours in water, without extra help by me and my blade!!!

I have not mentioned this observation before in this thread, but I have been quietly aware of it all along. It is a very reliable and ominous sign of failure for WEC embryo culture IME.

:frowning:

This is my hypothesis of what might be going on here:

My guess is that their trip in airmail cargo holds of jet airliners travelling thousands of feet in the air may have possibly subjected them to enough sub-zero cold to injure most of them. It is possible that in a population of these, a small fraction will have survived such physical insult, for whatever random or genetic reason.

I can almost guarantee that if these achenes were freshly picked, I would have seen a much increased germination percentage. I have noticed this discrepancy between fresh and dry+airmailed achene too any times now, to not mention it here.

This sort of damage seems to be totally random…some seeds are very “affected” whilst other seed in the same consignment may show lesser degrees of this “damage”…and in some crosses no such pattern of injury is apparent, in the same consignment. None of this surprises me any more. However it is totally fascinating to observe and speculate about, IMO.

I have learned to expect this sort of result, and just hope that the numbers I play with are large enough that one embryo is encountered which has resisted these insults, and hence proceeds to germination!

These four R.gigantea seeds will now be soaked for 3 days in tap water (water changed daily). In the first 24 hours of this 3 day soak, I will be removing their papery coverings.

Stay tuned for the embryo shots, and a written description of the embryos. I really hope one of these is a pearly white embryo which literally pops out of its testa covering (these are the most reliable predictors of a very viable embryo and a successful germination IME **[u].[/u]**

32

Well…

None of the four embryos that came out of the above seeds did so with ease…All had to be teased out of their papery coverings, there was no happy “popping out” of embryos, it is too bad!!! This alone signals unfavorable outcomes, yet again!

Of the four seeds, one was definitely dead and useless inside, another had an embryo so waxy and brittle that it fractured badly with attempts to release it from its papery coverings.

Here are the remaining two embryos of those original four, which showed some prospect of germination:



The smaller one of these two unfortunately showed a very distinct waxiness which extended from the tip of its radicle to almost the entire span of its cotyledons…oddly the rounded tips of its cotyledons remained pearly white… it is totally impossible to demonstrate such subtle color contrast details in these webcam pix.

The fatter of these two caused me a lot of grief when I tried to remove its papery coveings, especially at the radicle tip, where there was a persistent “cone” of testa tissue that would not free itself without the blade getting dangerously close to the radicle. No easy “popping out” of the embryo here, either. It was lucky that this tight cone of papery covering over the radicle finally dislodged without damage to the radicle!!!

This fatter embryo seemed to have some patchy waxiness around the curved tips of its cotyledons, however the all-important radicle tip and nearby cotyledon appeard reasonably white.

I give the fatter one a 50% chance of germination and the smaller one 5% chance of germination in the WEC run, based soley on these initial but very important observations.

For now, both of these continue their 3 day water soak. Perhaps the samller one could do with a longer soaking time???

Stay tuned.

33

Here are the 4 embryos after a 3 day water soaking (compare to pic taken of these, mon 21st feb, further up this thread):



Although these 4 look pearly white in the picture, in bright light they do have some patchy “waxy marbling” surface markings. That is, in my opinion they are damaged to some extent due to the drying and possibly low temperatures during air travel.

They are now sown in this drinking glass+ wetted perlite+cling wrap+elastic band set up:



I’ll undo the cling wrap once daily and freshen their air up, and mist them as well as the underside of the cling wrap at that time.

34

UPDATE:

The last two embryos discussed in the postings above were both sown today, (two and a half days total water soaking time). Both started to open cotyledons, the one that had extensive waxy changes involving the radicle/cotyledon areas still showed the same degree of dense waxiness. I decided not to soak it any longer, as I a have no hard evidence this would be of any proven benefit…this is all “guessing games” to be honest.

The two earlier embryos I discussed further up which had greened up and which showed radicle blunting (resistance to develop a rootlet), were sown a few days back in a medium consisting of:

commercial seedling mix : perlite (1:1).

Here they are as of a few minutes ago:

Seedling #1:



This was the least developed of the two “radicle blunters” at the time of transfer from their glass of perlite. At the time of its transfer, both its cotyledons were very devoid of chlorophyl, and there was no sign of true leaf emergence. However, since the last 24 hrs, a true leaf is forming, which to me means very positive things are happening to it.

Seedling #2:

This one had some signs of true leaftlet formation at the time of its transfer, and the true leaf has developed further, to this:



Based on these early results, there is now a very good chance this batch of R.gigantea seed will produce viable plants. Of course, time will tell.

35

It is becoming quite clear that this whole group of R.gigantea embryos are slow to germinate in the embryo culture environment, compared to some other embryos I have seen over the years.

I think I last counted 6 R.gigantea embryos now left, all growing in the one drinking glass (containing wet perlite+wetted cling wrap). All have swollen up, including the one with the radicle end which was densely waxed up!!

One of the embryos has green cotyledons whose rounded tips seem chlorophyl deficient (whitened)…I wonder does this pattern of zones of “whitening” (of the otherwise greening embryo) correlate with the waxy zones???..If this is so, one would expect the embryo described above, which had dense waxy changes to most of its surface (including all the radicle and the adjacent cotyledons) to show a whitening of these same regions as it “greens up”, and I would expect these areas of white to rot…in this case since the areas involved are in the critical radicle area, I would also expect the entire embryo to rot away and not germinate…I am particularly interested to see how this embryo develops, I have marked its position so I can track its development, watching out for this pattern… hmmmmm… interesting!

Some of the others in the group are going a yellow-lime color, a very good sign.

The fat one of the two pictured further up looks to have at least one extra cotyledon. So with that one, I am predicting a higher chance of “establishment troubles” if it ever progresses to become a seedling.

It is all fascinating to watch unfold!

36

I should add, of the two embryo-seedlings that seemed to show radicle blunting, the more developed of these two is now growing a second true leaf and is showing the beginings of a stem, whilst the lesser developed one declined and was eliminated yesterday…it was not exactly dead, but looked hopelessly “frozen and hardened”.

It looks as though there is now definitely one R.gigantea seedling happily growing away, out of all this fuss!

Hurray!

37

Here is R.gigantea seedling #2 as seen today:


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Here are the remaining R.gigantea embryos in perlite, as seen a few minutes ago:

…this one is a tricotyledon :frowning:



This one is also a tricotyledon :frowning: :frowning: :frowning:



Another embryo:



Another embryo:



Another embryo…this one is a little more greened up, but it has those worrying whitened zones (such whitened zones in previous attempts have tended to rot away rather than green up as the embryo-seedling has developed further along):



Sooooo… in summary, I am noticing that after almost one week in the wetted perlite, there is a pronounced lack of chlorophyl synthesis in all but one of them…This pattern is definitely out of the usual norm, IME…could this lack of chlorophyl synthesis/white zonation correlate with the waxy zonations noted on these as newly extracted embryos???..Finally, the embryo shown below is the one I wrote about yesterday, which had nearly 100% of its embryo surface being very densely waxy (except the rounded tips of its cotyledons which were pearly white).

This embryo will be the best example I have had so far to use to test whether or not dense waxiness appearing on the surface of some newly extracted dry rose embryos correlates with white zones in their greening-up phase, which in turn correlates with zones of necrosis/rot in the seedling phase:

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Verrrrryyyyy slowwww embryo progress, but there are no signs of decline or death either!! LOL!!!

Could this slow development of these R.gigantea embryos be a hallmark for the R.gigantea line??..or does it have something to do with how this species seed responds to extreme drying and high altitude air travel??

Hopefully one day one of these embryos will become a mature plant, and bear fruits and seeds, so I can revisit this, and compare the dry seed and the freshly picked seed embryo responses to WEC.

The partially greened up one was potted up yesterday, it has a tiny true leaf emerging and did have a rootlet forming. As predicted, its white leafy tip zones are slowly dying off as its “apical meristem” region (which is the green zone in this case) takes over. This white zoning is weird!

One of the tricotyledons was potted up today as it was greening and had the start of a definite rootlet growing.

The remaining four others continue to slowly develop, but are still too immature to consider them ok for transfer to a pot. The very waxy one is not dead!!

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Seedling#3

I potted this one up yesterday (note how the coltyledon tips are refusing to green up):



Seedling#4

Here is one of the two tricots… it was potted up this morning. Note one of the cotyledons totally refusing to green up…my guess is that this whitened cotyledon will remain non-functional, and die off.

LOL!!


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