Hi Larry,
interesting thoughts, thank you for sharing, for me it is thought provoking!
I am still pondering the many possible reasons about this run failing.
One other possibility I have so far not mentioned in the writings here, is the possibility that the hips from which these embryos came from in the above runs were picked too unripe (they were still green and it is mid-summer, so they had some ways to go if left on the plant).
The gigantea achenes I am soon expecting to receive apparently come from fully ripened and colored hips which had dropped spontaneously from a chinese R.gigantea mother plant in mid-winter (France).
When/if I receive this seed, I’m now thinking to re-try the embryo run in case there is any difference in germination rate to do with the ripeness of the hips (green hips picked off the plant in mid-summer versus colored hips which have dropped off the plant in mid-winter).
I will this time also increase the water imbibition time from overnight (as done in the two runs here, further up) to at least two days to see if that helps embryo responses.
Also if there is enough material, I’ll try and do a parallel “seed run” to the “bare embryo run” using seed with woody pericarp totally removed (but testa deliberately retained), to see if there is any noticeable difference in the final germination rate.
For those interested in such things, here is what R.gigantea XOP 2 seeds (hard pericarp excised) and 1 embryo (testa excised) actually look like, fresh out of their hips (no cold stratification):
Scale: 1 gap = 1/16 inch.
[attachment 308 Picture5.jpg]
OK.
I actually have the two intact seeds shown above (no hard pericarps) and 2 bare embryos (one was not pictured), all in water immersion right now. All four came from the same green hips, picked mid-summer here in Australia.
Since my damp peat moss trial stuffed up, I have to search for better ways.
I hate paper toweling, but I do love the zip-lock baggies others (and I) use for embryo work and seed stratification.
I’ll try this:
After these four have undregone a two day water soaking, (and if there are no sprouters in the water at that time), I’ll transfer what has not sprouted in the glass of tap water into an unused/new zip-lock baggy, sprinkle a bit of water inside, drain off any water that runs out, and seal the baggy off and place it in the fridge. The fridge temperature dial will be set @ roughly the half-way mark.
Then I’ll transfer the baggy out of the fridge periodically, like say once a week (e.g every Sunday) for 24 hrs, then put it all back into the fridge.
Whenever/if I see sprouters in the baggy, I’ll sow them in my usual germination mix.
I’ll repeat the same protocol with the seed I am hoping to receive from France soon, (as a contrast that seed was picked mid-winter from fully colored hips).
I’ll post results when it is all over, to save lots of unnecessary writing here.
George, does your water have chloramine or chlorine in it? The chloramine is really bad for plant roots, and maybe the other parts too. The chloramine is really hard to get rid of…
Hi Larry,
Thanks for the heads up on this possibility.
I have thought of using distilled water for these experiments to be able to standardise things a little, I know that tap water is anything but a pure compound (H20).
It is a great point that you raise.
Here is a publication from the local authority responsible for such quality checks, which does make mention of chlorine and chloramine: http://www.sydneywater.com.au/Publications/Factsheets/WaterQualityTestingAndReporting.pdf.
I don’t now if this is reassuring, however I would like to add that so far after the two day water soak these embryos are looking definitely swollen and clefts did appear between the cotyledons, there were no blemishes or discoloration to alert me to any premature demise or other bad process going on. I am not too worried about the local water, because to go back in time a bit, I also still cannot get over the fact that a couple years back, I was able to germinate a percentage of R.multifloraXOP seed in a glass of this local tap water, even after the 50 day immersion mark!!! (Of course I don’t recommend anyone seriously trying this other than if they want to have some weird fun on unwanted R.multiflora seed).
I am almost convinced out of sheer gut feeling, that leaving the seed coats intact and not removing the testa is the way to go here. The bare embryos appear soooo fragile, yet their siblings with their seed coat intact (no hard pericarp) really look safe and snug in the baggy.
BTW…
In the mean time, I have been meaning to ask for a while, but never got round to it…
Has anyone on the forum germinated R.gigantea achenes, and what was their experience with that?
OK.
already I can see that the embryos in the zip-lock baggy are drying up as they are stuck to one side of the plastic, I am almost 100% sure this latest embryo side of this trial is going to fail, and fast.
I’ll modify and place the embryos on wet paper in the baggy, and the seeds (pericarps removed) I will leave as they currently are, left in a separate baggy just with a few drops of water and no paper.
George, Sydney is an interesting place in terms of water treatment. Depnds which system you are on whether you avhe chlorine or chloramine. But I would go for boiled “clean collected” rainwater, or bottled distilled myself, if I were doing tests of germination.
Hi Larry,
In that case, I’ll use distilled water for the French gigantea seed (hasn’t arrived yet).
For those interested in such things, here is the appearance of some of the dry R.gigantea achenes I just received from France (derived from fully colored hips):
SCALE: 1 gap = 1/16 inch.
[attachment 315 Picture1.jpg]
Pictured below are a few of the extracted seeds from some of the above achenes (woody pericarps removed).
This seed has a darker brown color compared to the very light tan/grey color of the seed that came from the green hips (see further up). The seed coats of these mature brown colored seeds is considerably thinner than the seed coats from the seeds out of the green hips.
(Note a small “window” of the brown seed coat has been sliced off the seed second from the left, revealing the white color of the embryo within it):
[attachment 316 Picture5.jpg]
After an overnight soaking in demineralized water, I kept all those that had intact seed coats (N=6) and converted those with partially torn seed coats into bare embryos (N=5).
It is very interesting for me to observe that none of these embryos had waxy hard brittle appearances (unlike the Indian seed which was all waxy hard and brittle). This is an extremely positive sign because it denotes greater chances of success in the EC run (in my experience waxy type embryos are very hard to get going in a span of 1-2 weeks of the usual EC, and have usually failed me in EC). It makes me think that the air travel had nothing to do with promoting any “waxy changes” in the Indian seed.
This seed had only been dried for a couple weeks (max) from time of removal from the mature hips to its delivery to me in a paper wrapping (not in a plastic baggy). In this case, 2 weeks of air drying and travel in air-cargo holds did absolutely nothing to promote any “waxy changes” in this particular seed. All of it looks very healthy indeed!
I have no idea what causes embryos to appear waxy hard and brittle, and these changes are certainly linked to very poor outcomes in the usual 1-2 week span EC I have run. If I were to have a guess, I think hard waxy brittle embryos are those that have been subjected to some form of extreme drying damage, to the point they are very water repellant (just a guess).
Modification
As much as I don’t care for it, it looks like l better also include wetted paper toweling for the bare seeds (pericarps removed) arm, because a few drops of water in the baggy instead of wet paper toweling seems to be giving either too much or too little water to the 2 seeds already in the fridge, depending on where they sit in relation to the water drops. Duuugh!!!
O:)
Hi George,
I’ve been meaning to ask how you remove the seeds from the hard pericarp without destroying the embryo in the process. I know how we do it for peaches: A pair of double cut pruning shears lined up with the stem end of the suture, a quick snap and it’s out. But, I don’t see that working for rose seed!
Hi Natalie,
I am pretty sure you are right about the pruning shears, although I must admit I did try a few light taps with a hammer on the gigantea seed and that was no good either…duuuugh!!! … (FYI, the hammer trick works for peaches and for olives if done delicately and on the suture line, but you risk hammering your finger/thumb, albeit ever soooo delicately, as well…OUCH!!).
Have you ever done embryo extraction (EE) work on rose achenes?
If not, it is probably best to try Don Holeman’s fab method as published on the RHA home page articles on Rose Hybridization /Simple Embryo Culture for Plant Breeders, here is the direct link to that great article, to save you time navigating:
http://www.rosebreeders.org/embryoculture.pdf.
To put a time perspective on myself, I started messing around with rose embryos a few years before getting to know about the existence of RHA, and therefore about Don’s work. As a consequence of that, I had already developed an alternative method of EE, which worked for me, and which I keep using as I am so used to it. If you really want to know how I do it, email me and I’ll try and explain in detail, to save writing here (this thread is already getting too long LOL).
:O)
UPDATE:
The (green hip-derived) embryos look hopeless on inspection today, and therefore were discarded.
The (colored hip-derived ) embryos are approx. 1 week behind in terms of cold storage, but some are starting to show some browning of the root tips…I think this is the start of their end, but lets see.
On the other hand, both batches of the seeds with intact papery coats (hard shells removed) are mostly ok…however I think one or two out of the bunch from France look to be dying (these had developed a little mold whilst immersing in the the 2 day water soaking, so I never expected them to be ok, however I included them anyways out of curiosity).
Interestingly, the 2 seeds derived from the green hips have changed color from a tan/grey to a mahogany, which is the same color as that of the seed which came from the colored (fully matured) hips. Therefore, I am guessing / hoping is actually a positive sign.
This one was pictured today, after it and its sibs just finished their one cycle of 24 hrs out of the fridge per week (room temps ~ 70-80F).
[attachment 348 Picture7.jpg]
I’ll leave the one pictured above at room temp indefinitely now, in its own individual baggy, using wetted paper (wetted with the demineralized water). Maybe it has lost enough dormancy to move ahead (it will be very obvious in a day or two what the answer to that might be, to be sure).
As for its other embryo sibs, two were dead today and two remain, one looks a bit like the one in this picture but without a rootlet yet (its root tip is brown), the other looks like its going to heaven but it was given every chance and those two are back in the fridge for the next six days before being taken out for one day in room temp.
NOTE: This case is now 19 days in embryo culture. As I never used the fridge to culture embryos in previous attempts over the years, I was never able to sustain embryos for more that about 2 weeks before any remaining unsprouted ones perished.
Now the embryo and true seed (pericarps removed) cultures are past the three week mark.
There is only one remaining embryo alive. That embryo looks also to be getting some brown marks which I read to mean some chemical/physical reaction that predicts its death (just a guess).
Here is a picture of the remaining embryo as seen through the plastic baggy:
[attachment 356 Picture8.jpg]
All that seems realisitcally left are the intact seeds with hard pericarps removed (none of those have sprouted yet, most appear fine to the eye, though I have not opened their baggies to feel them in case any of them squash easily).
Today is a few days short of four weeks since the French seeds (harvested from fully colored hips in mid winter) were put into the baggies and fridge (pericarps removed).
I just noticed two of them are germinating (here is how they look through the baggy today):
[attachment 360 Picture81.jpg]
[attachment 361 Picture91.jpg]