Hi all.
Jukka, I love the new thread/discussion you opened yesterday with references to some ideas I have put forward in my very busy and cumbersome original embryo thread (refer to- http://www.rosehybridizers.org/forum/message.php?topid=23111#23111).
With your permission Jukka, and with the collective permission saught of readers here, I would now like to copy/paste all the conversations that have so far been posted on your thread since it was started yesterday, onto this new thread here, which has a header title to it that I feel describes my current embryo culture method best, and which you would not have been aware of at the time you posted your title to your thread yesterday…Your title is your prerogative, and it is fine, however with your permission, I feel it just might be very clear right from the start of this discussion, that Jar Embryo Culture is an obsolete method that I have upgraded to “Water Embryo Culture (WEC)-2010 version”.
I don’t mean to be controlling or pedantic by doing this, as a matter of fact this idea has been prompted mainly because one prominent member of this forum has just emailed me to say that he was not even aware of all the embryo work I had posted, over 12 months worth, on the thread with the unfortunate and irrelevant title of “Can anyone identify this red climber” (http://www.rosehybridizers.org/forum/message.php?topid=23111#23111)…in effect the original irrelevant title of that very huge embryo thread, robbed that thread of very valuable potential member inputs…it is too bad, but I can now move on with your permission in this manner, knowing that for once I have entitled the work correctly for its content!!!
Thanks!!
So here below I have copy/pasted of all the discussion so far from Jukka’s intriguing thread which he started yesterday:
Posted by Jukka on Sat, Nov 20, 2010
Because the original thread on JAR EMBRYO CULTURE etc. has grown so long that it is difficult even to browse it, I start a new thread here and paste the description of the method by GEORGE, SYDNEY, AUSTRALIA. Hope you don’t mind, George!
Thanks, George, for posting your method! And to all others for making useful comments and suggestions. JEC has already revolutionized my very small-scale amateur hybridization. In my cold climate in Finland it’s hard to get hips ripening well and those few that do always show disappointingly poor germination. I was already giving up the whole of rose hybridization and pursue only rhododendron hybridization (which is FAR easier) when I came across your post about jar embryo culture. I immediately gave it a try and now, even though I’ve processed just a few dozen seeds, I have several small seedlings from two crosses (Duchesse de Montebello X John Davis, Golden celebration X Klaus Groth) growing on my window sill! I can’t wait to get seeds from my other crosses processed! I’m very excited about getting seedlings from my Alfred Colomb X Flammentanz cross. Hoping for some fiery reds!
So, thanks again, George!
Jukka
Helsinki
Finland
Quote from a previous post by George, Sydney, Australia:
"When I first started playing around with this idea of mine (early 2009), of course I was slicing into and squashing a lot of embryos. In order to get high percentage intact embryo extractions, I realised it was important to define the long axis of the seed with a few initial key slices. Sometimes it was easy to guess, other times a little more slicing was required to define the long axis orientation of the seed within the achene.
After you get the long axis defined in your head, you then slowly slice away at the hard achene in a direction along/parallel to the long axis of the seed, until you have removed enough achene to identify the cotyledon end of the seed. Now you can slowly extend the attack to remove the entire cotyledon end of the achene.
Soon enough you will end up with the radicle end of the seed buried into what is left of the radicle end of the achene. At this point, a few gentle wiggles on the seed with your box cutter blade usually dislodges the seed out. If it fails to dislodge easily, then a few more careful slices on the achene remnant should do the trick. Damage to the radicle is to be avoided at all costs, so this is the critical and most defining few cuts you will be making…(damage to the radicle spells embryo death).
I then place the seed with its seed coat into a glass of water for a while until it visibly hydrates a bit (it gets firmer and shinier). Sometimes if you are lucky, the seed coat has been sliced a little through the achene removal, and the embryo pops itself out through this slice in the glass of water…(this is very time saving when it happens!!!).
Most of the seeds however have a pretty much undamaged coat if you have been “good at it”, so they require extraction of the seed coat. In such seeds, I then proceed to remove the seed coat by making a very superficial side-ways slice at the cotyledon end of the seed coat, to just reveal the pearly white surface of the embryo underneath.
I then slowly nick away and pull away the seed coat with the box cutter blade, always avoiding getting anywhere near the radicle end. The seed must be handled delicately yet purposefully with the fingers of one hand, whilst using the cutter with the other hand to unfurl/peel away the seed coat layer. Much care is required to not squash the embryo here with the fingers holding it in position. Sometimes the embryo pops out at a critical point of losing most of its seed coat. However, in other cases it does not release itself easily, in which case I do not remove any more seed coat if it is nearing the radicle… Instead, I immerse the semi-extracted embryo into a glass of water until it finally pops itself out.
An advantage to using the box-cutter is that you can slice at short/acute angles which gives you very good control of where you direct the forces, to avoid crushing the embryo underneath. The knife must be very sharp of course, otherwise you will be forcing it too much to make the cuts, and thus risking blunt force injuries to the embryo within."
Posted by Jeff Stover [email] on Sat, Nov 20, 2010
There’s a PDF booklet that Don Holeman put together about JEC. It came out about the same time the JEC thread came out. I’ve been through it several times and find it informative and easy to follow.
If you missed it here’s the link;
http://www.rosehybridizers.org/embryoculture.pdf
Posted by Jukka on Sat, Nov 20, 2010
Thanks Jeff!
Posted by George Varden (Australia, zone 10) on Sat, Nov 20, 2010
Jukka, its great if this has helped you!
Posted by George Varden (Australia, zone 10) on Sat, Nov 20, 2010
Jukka, this is the “Water Embryo Culture-2010 version”, which is the final refinement of the original Jar Embryo Culture method. By experimenting over the last 12 months+, I have found that this “Water Embryo Culture” method is superior to my initial use of Jar Embryo Culture.
There are two steps involved:
- Embryo extraction:
Using a box-cutter knife (the detail has correctly been copy/pasted further up this thread):
Knives have to be very sharp to be effective at cutting, (rather than squashing LOL). Accept the fact you can cut yourself, and things can fly around in the air…be careful to avoid injuries/damages!
2.Sprouting the extracted embryo in a jar of water:
By placing the embryo in a glass jar (eg.baby food jar) partially filled with room temperature tap water, and letting it immerse for as long as it takes to start seeing radicle elongation starting. This is done in a dark cupboard, away from light.
So, you start with this:
and days/weeks later you end up with this:
Once a radicle begins to elongate, as seen in the last picture, the embryo is then sowed as though it was a seed (rose achene), in the usual seed germinating medium…and must be kept ALWAYS moist until germination occurs (germination can take around 1-2 weeks typically).
There are usually a few embryos that resist sprouting, and once these start to discolor, just throw them out. I guess these embryos may have higher than average chemical dormancy in them, or have been damaged either by a badly done extraction or by some other injury.
Posted by Jim Sproul (zone 9) [email] on Sat, Nov 20, 2010
Hi Jukka,
This was a great idea to bring the subject into it’s own thread. I have read some of the material with interest, but this will be much easier to follow.
Hi George,
I am sure that you may have mentioned this in the other post, but have you found significant differences between traditional germinations and embryo extraction germinations of poor germinators? I have some seedlings that are intermediates in my breeding that set hips excellently, but the germination rates were found to be horrendous. I have abandoned using them, however, if germination is significantly improved, I may take the time to do pollinations again followed by embryo culture. Thanks!
Jim Sproul
Posted by George Varden (Australia, zone 10) on Sun, Nov 21, 2010
Hi Jim.
What a pivotal question!
There are great dangers in me delving into generalizations here, however I have a few ideas I would love to share regarding this challenging situation you bring up:
In the first instance, if it were me, I would make a small number of these crosses say in order to obtain ~50 achenes to use for sampling purposes. I would open these achenes up using embryo EXTRACTION, and see what lurcks inside! Then I would embryo culture all of the normal looking embryos, on this small scale, and see if there is any really useful improvement in the germination rate. If there is, why then certainly I would consider embryo work on this cross, on a larger scale.
For example, by using the embryo EXTRACTION step alone on these 50 achenes, one may discover the following hypotheticals:
A significant population of live healthy embryos in the sample (healthy = pearly white, shiny, slippery) . Here one might suspect some sort of true “physical embryo entrapment syndrome” of a potentially very fertile cross. I have no idea how common this sort of phenomenon is in the real world of rose embryos.
So, if this were the real case finding, and it were me, I would proceed to embryo CULTURE all normal looking extracted embryos. If the % germination was significantly better, well there is your answer! It would be a GO for embryo culture, in theory, on a larger scale.
Next-to-zero healthy embryos are found, lots of empty seed sacs, black seeds, transluscent soft non-viable seeds…in this case this cross shows very low fertility.
So, if this was what I found, then I would still embryo CULTURE any normal looking embryos and again calculate the final germination rate. If it is similar to the usual near-zero rate, then I would certainly not bother with embryo culture on a larger scale. This would indicate a very low fertility situation. If however the germination rate was much higher than the usual near-zero rate, then maybe these very few embryos that were present were also being trapped in tight sutures…a double whammy!! In that case it is a GO for embryo culture, in theory, on a larger scale.
I say “in theory” because in the real practical world, the relatively massive amount of time and energy that it takes to produce even ONE seedling by embryo culture, compared to simply sowing one achene to get the same one seedling, means that the larger the number of achenes you have to process for embryo culture, the more disproportionate amount of time and energy you will have to put into the whole thing. It can have the potential to become a very taxing venture, depending on your skills and motivations, so beware, and, take care!
