(clinophylla x bracteata) x op.
2nd germination potted on day 4 of jar culture, as seen today after 1 day of growing in the pot.
(clinophylla x bracteata) x op.
3rd germination potted on day 4 of jar culture, as seen today after 1 day of growing in the pot.
(clinophylla x bracteata) x op.
4th germination potted today, day 5 of jar culture
(clinophylla x bracteata) x op.
5th germination potted today, day 5 of jar culture
This posting is an answer to a private email query I received recently from an RHA forum contributor, who was having some sort of ādryingā troubles with his embryo work. I am not sure what sort of embryo culture he was utilising, and I am not sure at what stage the embryos were drying out, however I thought I would post my response here, to also benefit other readers here who may be interested in jar embryo culture.
Here goesā¦
I routinely immerse all my embryos these days as soon as they are extracted into a clean glass of cool tap water for at least 24hrs (I make sure they have sunk to the bottom). Then I submit them to the jar culture. Maybe I should be keeping them in water even longer, until they actually start to open their cotyledons, or show other definite signs of growth (I have done this and have been quite successful with the embryos in water for 2 or even 3 days).
With this jar embryo culture method, you need to keep an eye out especially for re-emerging dehydration, once the embryos are in the jar. It is not necessarily that the jar is not maintaining humidity, it has often more to do with the quality of the embryos you have in front of you, and their ability to maintain adequate water balance without help from you.
Some embryos just donāt maintain a good water balance in their first few days in culture. These need water resuscitation, and this can take several rounds until they start to independently maintain their own water balance.
If any of the embryos seem to have no āhaloā of water around them on the side of the fogged glass, you resuscitate them by simply taking them out of the jar, and immerse them into a clean glass of cool water probably overnight, then place them again on the side of the fogged jar and look for a halo of water to develop around them (have a look at some of the pictures above to see what I mean about this āhaloā of water). If this does not happen, sink them in tap water for longer, yes longer. A dead embryo will eventually just disintegrate, get rid of it, it was never any good and will only become a nidus for fungi to feast upon.
Keep the interior of the jar fogged up at all times, and re-fog the jar any time you think it is losing the fogging by exhaling into it a few timesā¦To be honest I might have to do this once every day, sometimes more sometimes less frequently, it is not a big deal. Of course keep the lid sealed well, and ensure there is a water line on the bottom of the jar. Keep about 1/2ā gap between the water line and the lowest embryo.
The water halo sign is a subtle yet fairly reliable sign of good embryo hydration and health.
Oh, and by the way, the reason I am emphasising ācool waterā, is because tap water can be hotā¦LOL (eg. on a hot day, or the hot water was running just before you got to the tap againā¦Please test the tap water by letting it run on your hands for a while until you are sure it runs consistently cool, before using it on the embryos. You donāt want to boil them!!
Hope this helps answer your email question, best of luck!
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George,
You really ought to collate all this great information into a followup article on embryo culture for the RHA Newsletter. This could be presented as one personās practical application of Donās technique presented last year. I think many people will find this very meaningful.
Paul
Paulās comments here have prompted me to now explain a little about the origins of the jar culture/box cutter ideas I have posted all over this thread.
It was sheer coincidence that Don was just about to release his RHA document relating to his very involved embryo work, not long after I started chatting about the jar culture/box cutter thing on this thread. I had only just become familiar enough with this RHA forum, and was just starting to even get a grip on who was who, (if you know what I mean?!)
I had stumbled upon this RHA forum by chance only 3 or so months prior, and I had no idea an RHA member (Don) had been so involved in all this embryo work to the point of an imminent publicationā¦ Such things happen in life, its ok.
So I started to reveal my method here, and then Don asked me questions about my method, and then kindly sent me a copy of his work which was about to be published.
It immediately fascinated me to see the different angles of attackā¦very cool and quite a funny coincidence.
What I have done here is just out of my own mind, it is NOT based on any scientific readings/research papersā¦and it seemed to work. The whole jar/box-cutter embryo idea came to my mind some time ago now, when I had not even thought to dabble in roses but was dabbling with olive pits. I wanted to get olive pits to germinate in less than the 1-2 years they normally require, as I wanted a few olive seedling rootstocks to use to bud varieties of olive that are extremely unreliable to root as cuttings. My intuition told me to start cutting the pit with the box cutter knifeā¦ BAD IDEAā¦Slowly over the seasons, I realised that with olive pits I could do away with the box-cutter (at least for the step of removing the seed from within the pit) and instead apply a small series of light hammer taps to the suture line, and this usually results in fast successful release of the olive seed out of the hard pit.
NOWā¦ a long time later, I seem to have have developed a fixation in rose breeding, so I just modified the olive pit idea to the rose achene (without the hammer step LOL).
Look I am the sort that just likes to muck around experimenting trying to find practical solutions to stuff that bugs me. A lot of us do this without even thinkingā¦it IS FUN!
Speaking of fun, here is a picture comparing a 6 day old rose embryo (left ) to a 4 week old olive embryo (right), both still in need of more jar culture.
I would also like to say here, that this jar culture thing remains a āwork in progressā.
I am currently doing a few studies to see if I can get the same sort of percentage germinations from good embryos cultured in a jar versus good embryos simply planted in a germination medium (like commercial seed raising mix you buy at the shop). I think the main ātrickā here will be to tweek the water balance thing (too much water and they rot, too little water and they dehydrate and die).
It is quite easy to get SOME embryos to germinate if you plant them directly seed raising mix, that I already know. I just want to see how I can get equal results to the jarā¦That would be soooo much easier, agree?
Anyway, I will keep those of you that are interested in this stuff updated like I just have, on this thread.
My ultimate aim here is to have fun sharing with people like you who love roses, anything that might lighten up your day, even if it amounts to you just having a quiet giggle with yourself, after reading stuff like this!
Hi.
I want to show you some more pictures captured by my trusty little webcam, which by the way is an invaluable tool in my embryo culture work.
Why invaluable? wellā¦you can unclip the cam from the frame of your computer screen and it becomes a mobile high quality video magnifierā¦ It provides immediate live magnification of things you could never hope to see with your eye, in their very early manifestationsā¦the earlier you detect a deterioration, the more chance you stand of being successful in your corrective action. I might do this once in the morning and once at night, to monitor subtle changes in progress.
Now back to the pictures I wanna show here.
This (clinophylla xop) embryo is currently in water resuscitationā¦it looks poor qaulity, it is 6 days in jar cultureā¦a few days ago it was removed from its original colony and isolated in what I call the āquarantine jarā . Here it is receiving water resuscitation. Basically the quarantine jar is exactly the same jar as for embryo culture, only instead of the embryos clinging to the side of the fogged jar, away from the water line beneath them, in the case of the quarantine jar the embryos are simply sunk to the bottom of the water line, until they either open cotyledons and plump up, or die, whence they are discarded, one by one.
Jar embryo culture does have some versatility, to be sure.
Here is a second poor quality embryo (clinophylla x op) undergoing water resuscition in the quarantine jar (day 6 jar culture)ā¦I can see a slight blemish on the cotyledon, this is worrying, it is probably a sign of death, but it might be artefact/shadowingā¦Iāll soon know, but I must give it every chance, as this exotic species has come all the way from India!
Here is a third embryo (clinophylla x op, day 6 jar culture) in the quarantine jar undergoing water resuscitation.
In this case with the marvelous help of the webcam magnification, I can see subtle signs of cotyledon opening, so this one is in a better condition than its other two fellows pictured immediately above this entry.
This embryo (clinophylla x op, 6 days jar culture) was in water resuscitation for 2 days. It initially looked like the first two poor quality embryos of this series of four pictures, but it opened its cotyledons, and got shinier and bulkier (put on wieght) and maintains a good water halo in the fogged jarā¦It now stands a good chance of germinating crosses fingers
So, never give up on them, especially if their provenance happens to be, well, āpreciousā (in this case India>Viru>Simonā¦LOL).
A NOTE TO THOSE READING THIS WHO ARE PERHAPS NEW TO THIS THREAD OR WHO MAY BE BEGINNERS IN ROSE BREEDING.
One RHA forum reader emailed me privately yesterday, and decided that all this embryo stuff was not for him this seasonā¦of course he was absolutely correct about that!
It is only meant to be used in rare cases where seed has some important breeding potential and ALSO known to be impossible to germinate in the usual fashion for rose seed (ie. by sowing after some sort of stratification, in most cases).
In my case here, I am demonstrating refinements of a specialised system, so I use whatever seed I have. This has nothing to do with normal germinating practice for rose seed.
I am sounding like a broken record about this, however the email I got recently compelled me to write this, just to help clear up any confusion some fresh beginners may have about the role of jar embryo culture.
TROUBLE WITH CLINOPHYLLA X OP in embryo jar cultureā¦
I was right, there IS fungus in one of the clinophylla x op embryos!
In fact 12 hours ago the webcam revealed a few tiny fungal filaments, but I decided to wait and see if this was fungus or just some webcam glare artefactā¦ there is no doubt nowā¦ To put the picture below into some scale, I can barely just see these filaments with my own eyes.
Now, I had been suspicious about fungus right from the time these showed abnormally slow growth (see discussions above), so at least I have been proactively chasing this covert little trouble makerā¦(I am trying to make myself feel better about thisā¦LOL).
I must deal with this, FAST.
I already know that directly planting good quality fresh embryos which have been pre-immersed in water for 24-48hrs has yielded germinations in the past for me (germination rate 10%-100%).
My instinct now tells me to immediately plant each of these embryos in its own individual pot, into commercial seed raising mix, and keep the whole thing on the dry side of dampā¦(maybe pre-wet the medium, then place the rinsed embryos on top, and then throw a little dried medium over the embryos to cover them, and simply mist-spray the surface, as required, to keep the surface just moist.
BTW, expected temperature maxima for where I live are 90F for today, and 102F for tomorrow (relative humidity hovering between 50% - 80%).
I donāt think that the weather is quite on my side, either!
Stay tunedā¦LOL
Molds tend to be opportunistic rather than pathogenic - they grow on dead or dying tissue. Sometimes part of a cotyledon can be dead while other parts are alive and green-up. Thatās what your photo seems to show.
Reducing the temperature can help. 55-65 degrees F. is a good range to germinate rose embryos. In that range the bugs grow more slowly while the embryos grow pretty quickly.
Hydrogen peroxide helps too.
Hi Don.
In the time between ending my post here, and receiving your post, I hgad already planted the five embryos from that jar in fresh seed rasing mix. I first rinsed them in water and then lightly misted them with a hand sprayer to clean them up again.
Thanks for your posting here, it is interestingā¦the fungus looked gross anyway, I just couldnāt stand the sight of it to be honestā¦LOL.
I should have photographed the other four of this colony in the jar prior to planting, but well I was in a hurry this time!
Three of them had greening-up cotyledons, and some rootlet formation, but not quite enough to declare them āgerminatedāā¦ all five actually do look in good condition otherwise, even the one with this fungal colonisation looked quite robust and ābouncyā when lightly sprayed down with my little hand spray-mister.
There are three others wich are the worst performers, those are isolated in another jar system altogetherā¦one of them seems to be springing open its cotyledons, but the other two remain inactive, yet have developed opaqueness, from initial transluscencyā¦there is hope there, also.
Iāll put the pots in an insulated cooler box thing known in this country as an āeskyā, to try and keep it cool in this sizzling heat. Iām doing this indoors of course.
:0)
Soon enough Iāll provide a simple summary here of the achene numbers, the extraction success rate, my predictions of culture results (which I made at the time of extraction, based upon the embryo quality/appearance), and the actual culture results.
I have assigned these embryos code names.
The fungus affected one is 02-10-05. It will be most interesting to study it as a germinated seedling if it gets to that point, to see if there is any correlation between the embryo pathology, and the resulting seedling appearance.
BTW I do all these embryo transfers in and out of jars and such, by swirling the water in the jar to pick up all the embryos. Once in the water, you can just empty the water and the embryos out onto a flat saucer from the kitchen. If they donāt all come out, just add cool water to the side of the jar, and repeat. I have never damaged an embryo this way, ever.
Then I use the flat end of a knife with a rounded end (eg. butter knife) to gently coax each embryo onto it, for transter to the germinating medium.
PRELIMINARY SUMMARY OF TWO PARALLEL JAR EMBRYO CULTURES:
ACHENE CROSS ā01-10ā: (Clinophylla x Bracteata)xop
12 dried achenes were received and processed Jan 12, 2010:
All 12 embryos were extracted undamaged (using a box-cutter knife)ā¦1 was gelatinised and discarded, 5 were totally dried beyond recognition and discarded.
6 remaining embryos were immersed in waterā¦(all showed excellent pearly sheen, and developed bulking-up and/or opening cotyledons, after 24hrs of water immersion).
PREDICTED JAR CULTURE RESULT: 6 germinations were predicted at the time this jar culture was started, based on this data.
ACHENE CROSS ā02-10ā: Clinophylla x op
20 dried achenes were received and processed Jan 12, 2010:
6 embryos were damaged from the extraction procedure (box cutter knife), and discarded. All 20 achenes were very dense, very difficult to open up, and the embryos were comparatively tiny, spindly and very fragile to handle.
14 remaining embryos were immersed in water (2 developed only moderate sheen, 12 remained dull or even partially transluscent after 48hrs water immersion).
PREDICTED JAR CULTURE RESULT: 2 germinations were predicted at the time this jar culture was started, based on this data.
My next entry will give the actual jar culture results (may take up to one week from now).