Don... extracted some embryos today :)

You’d be proud of me today Don :slight_smile: I took that little bag of OP ‘Baby Love’ seeds you sent me and cracked all 40 open and extracted 14 viable embryos… struggled with removing the testa more than the embryos this time (so must be getting better at it) and have them bagged up and on their way :slight_smile: On another note… I removed the 50 OP ‘Westerland’ seeds you sent me from the fermenting tomato pulp the other day (they were in there 2 weeks and the top had gone very mouldy)… and there has been a pretty dramatic change to the appearance of the achene. They look like they have been sand blasted and with a little pressure from my fingers on either side of one of the achenes (one that looked especially ‘eaten’) I was able to pop the two halves of the achene open. So this batch of 50 will now be split into two groups of 25 and the remaining lot will be split into two groups of 25 and I’ll sow one lot of 25 from the tomato pulp and one lot of the untreated now and then stratify the rest as normal and then sow them to see if there is any difference. I need to repeat the experiment and take photos this time and add it to my rose-blog. If you don’t mind I’ll photograph the embryos I did today and add them to my blog site to show sequential changes too.

I tried opening the OP Floradora seeds but destroyed them… they are a little smaller than the ‘Baby Love’ seeds and I’ll need to practice a bit more before tackling them again. I got one clinophylla x bracteata embryo out without mashing it… which is cool… so… I’m on my way with this embryo culture stuff :slight_smile:

Thanks again for you help :slight_smile:

Tasmania will never be the same.

I tried opening the OP Floradora seeds but destroyed them… they are a little smaller than the ‘Baby Love’ seeds

The fingernail clippers need to be very sharp when you try small seeds. Either get a new pair or sharpen the ones you have. I glue a bit of 500 grit silicon carbide sandpaper to a thin spatula using 5-minute epoxy to make a sharpening tool. Just touch up the cutting edges on both faces of the clippers.

“I glue a bit of 500 grit silicon carbide sandpaper to a thin spatula using 5-minute epoxy to make a sharpening tool”

Don, you are amazing.

I bought a new set of clippers (two of them… one huge one and one medium sized one… the smaller one is more useful), so they are still very sharp. The sharperning tool sounds good too… will get onto it :slight_smile: I find my biggest problem is physically holding the little seeds… how on earth do you hold them so you can still get the clippers in around them??? The big seeds are easy because I can get a good grip on them. Those little ‘Floradora’ seeds though are very frustrating. I’ve also found, however, that big seeds doesn’t always equate to easy extraction… gigantea was a PITB… weird angular seeds that are very thick with little embryo that were tightly wedged in even in very dry seeds. I took a photo the other day, Don, of an embryo I extracted from an immature hip. I was in a nursery and I really wanted to buy this rose… but it had RMV… so I decided to help them by ‘dead-heading’ it and removed the hip on the off chance it was mature enough… it wasn’t… the embryo was a strange translucent jelly-like material… quite cool really… will post it here when I have a bit of spare time to sit down and get it off the camera and PS it for publishing here…

how on earth do you hold them so you can still get the clippers in around them???

Very carefully :slight_smile:

It is important to have clean hands and to condition the skin on your hands, as strange as that may seem. I worked out a regimen that I follow rigidly:

Apply a small amount of Mary Kay Satinhands brand hand cream to your hands, especially the thumb and forefinger. Let it dry completely then wash the residue off your hands with warm water using Dial Hypoallergenic brand bar soap. Dry your hands with a clean cotton towel. This will leave your fingers with just the right texture to grip the seeds. The Mary Kay hand cream has also proved to be the best way to prevent cracking of the skin of my fingers due to repeated exposure to peroxide. Like much of my advice this may seem extreme and tedious but it took a long time and a lot of trial and error to get it right.

You’ll find that with time it will become second nature to you. Likewise, the use of the gadget becomes instinctive once you’ve mastered the basics of its use. BTW, I’d like you to send me a picture of yours - or maybe post one here - so I can see if it needs tweaking.

the embryo was a strange translucent jelly-like material… quite cool really

It is fascinating, please do post some pictures.

I’ve been learning a lot about how embryos develop and just what, exactly, it means for an embryo to become mature.

For instance, the pericarp develops to full size many weeks before the embryo and the testa does. The testa next grows into the space, or lumen, within the pericarp, with the tiny embryo (~ 1 mm in length) nestled at the bottom and the space above the embryo being filled out with a delicate matrix resembling wet tissue paper. As the embryo grows this matrix condenses and becomes a thin inner membrane that makes up the inner layer of the testa. The embryo gradually goes from being completely clear to milky white to fully opaque and white.

I have had some very limited success with germinating embryos as young as about 60 days, when the embryo is about 1.5 to 2 mm long and is starting to become white. There are some embros in culture now that are much younger than that but I don’t have much hope for their success because the peroxide treatment seems to overwhelm them. Next season I hope to work out a more gentle protocol that would allow us to figure out the lower limits of germinability.

I will eventually be writing about all of this, so keep track of your observations and keep taking photos, maybe we can collaborate on an article.

This is all very cool! Way to go Simon!

I wonder if a little Elmer’s Glue (or similar glue) on the end of a tooth pick could be attached to the side of the seed and let to dry, in order to make a “handle” for easier maneuvering? Be careful though, a tray of these might be mistaken for mini hors d’oeuvres!

Jim Sproul

Don… here’s the immature embryo:



You can see the suture line beginning to form and the testa forming form what looks like the radicle end upwards.

Also… here are images of the tomato pulp treated seeds:


\

Ok, that’s what I’m talking about! The white band running the length of the cotyledon is remarkable. I have not noticed it the immature embryos that I have excised. It charts new territory from a scientific perspective from what I can tell from the available literature. It clearly traces a vascular element. Some questions are -

  • what turns it white?

  • is it the first step in the transformation that I described earlier?

  • what happens next?

  • will it germinate?

On the tomato puree experiment, are you planning to let some seeds continue the treatment to see if the sutures soften to the point they can be easily pried apart?

Don… I think I may have been misleading in my interpretation of the white line. This embryo was inside a hardened achene and these fleshy white structures are what will become the cotyledons. The white line clearly is not a suture line. I think, as you have said, it is the beginning of some kind of vascular tissue that will develop into the midvein of the the cotyledon (though I was under the impression that the cotyledon didn’t actually have a midvein but to be honest I haven’t looked that closely at it). Another intersting observation is that since I put it in the bag to attempt its culture it has semi-hardened and gone brown. I don’t know whether it is rotting or whether exposure has fast-tracked the transformation of the testa (which looks like it is present but in the photo in an immature translucent form), as it is the same brown colour of more mature embryos that I have excised. Maybe it is in ‘survival mode’ rapidly transforming the testa in an attempt to protect the exposed embryo to give it at least some chance of survival???

On the tomato experiment… that sounds like a good idea… I should set up a series of samples left in for different periods of time to get some idea of the rate of ‘digestion’ of the surface of the achene. These were placed in when I mentioned it in a previous post… so what’s that? About 2 weeks ago??? I’ll have to go back and check. The effect has been pretty dramatic in that short time I think. It looks to have hollowed out the suture line a little as well. I’ll stick to the above procedure first and will try your suggestion with a different bunch of seeds when I go back to work in a few days. That way I can put them in a more controlled environment in our lab incubator. I think I’ll choose standard lab conditions temperature (as comonly used in Australia) of 25

Ah, so this is the embryo with testa, you didn’t excise the testa. The browning may be death coming on, some enzyme system in the testa working overtime because it’s being driven by the peroxide, carotenoids being produced, all or none of the above. You might see if you can find another similar one to play with and try to extract the embryo from the testa.

The problem you’ll have with the tomatoe paste is that it will itself eventually start to rot. If you can make fresh preparations and change them at some interval of a few days that might be better. It would refresh the enzymes and keep the bugs at bay. I wouldn’t worry too much about temperature control in the first iteration although it can’t hurt if you have the facilities. In your climate that probably means a refrigerator to maintain 25 degrees :slight_smile:

Thinking about this more… I reckon the white line is the suture line of the testa… The brown was embryo death… I tried to remove the testa tonight and it was brown all the way through.

I’m actually counting on the tomato decomposing to set up anaerobic conditions to encourage fermentation. Adding new material will be needed to keep the ‘plant’ going (like feeding a ginger beer culture :slight_smile: ). With this one, I added two tomatoes, one to start with and another after one week (then I got impatient and wanted to see what was going on :wink: ).

I’m actually counting on the tomato decomposing to set up anaerobic conditions to encourage fermentation

What’s the reason to want fermentation? You might want to think about the competing processes underway in the puree, and the up side and down side of each.

Here is my collection of rather random thoughts…

I was reading about the production of biofuels from the fermentation of grasses etc and found that in the production processes the material must first be pretreated and this was traditionally acheived by using acids to break the cellulose and lignin (they called it lignocellulose) down into their monomers subunits before subjecting it to microbial fermentation to produce ethanol. There are also some bacteria and fungi that have been found that will also do the same thing and that these were preferential to using acidic hydrolysis because they didn’t procude fermentation inhibitors that needed to be removed and so make the process less profitable. When pretreatment had been completed the remaining biomass was fermented by both bacterial and fungal processes in much the same way as it occurs in ruminants like cows and sheep and these sugar monomers were metabolised to prodcue ethanol as a byproduct. The inside of a tomato provides an acidic environment adn has resident microbes which I was thinking is how the tomato pretreats its seeds to begin the decomposition of the gelatinous placental tissue surrounding the tomato seed. Interestingly I also found that this gelatinous placental tissue in tomatoes is also very acidic… I assume to protect the seed from contamination. Then I found this abstract (http://www.cababstractsplus.org/abstracts/Abstract.aspx?AcNo=20043168574) which said that tomato pulp was fermented using Aspergillis niger for the purpose of enzyme production and during production cellulases, pectinases, and lipases were produced. Then I remembered that to save tomato seeds from your own fruit you needed to ferment them to break down the gelatinous placental tissue that surrounds them because it contains germination inhibitors (this gives a nice run-down of the process PLANTanswers: Plant Answers > Tomato Seed Extraction). In their natural habitat, wild tomatoes produce fruit that will fall to the ground if not eaten and the fruit will ferment and decompose leaving behind seeds that have been chemically and microbially scarified and ready to germinate. Similarly if eaten seeds are able to pass through the digestive systems of most animals being expelled sans the gelatinous coating containing the germination inhibitors (as all the cherry tomato plants that pop up around here bare testimony too LOL). Then Larry said that old tomatoes produce pectinases which reminded me of the above… The tomato seeds that came out the other end of the 2 week process looked VERY much ‘over processed’… where as the rose seeds came out with the outer coating (which looks waxy in a lot of cases), stripped off. By replenishing the metaboliseable material the process continued for the whole 2 week period whereas sources suggest that otherwise it would stop after 4-5 days.

So the hypotheses I kind of roughly came to were that by mixing the rose seeds in with the tomato pulp and encouraging microbial action followed by fermentation I could encourage the microbes to digest the coating of the rose achenes if I could keep the process going by providing fresh tomato pulp every 4 or 5 days. My next hypothesis I wanted to test was that without this out coating the germination inhibitors could be leached out more effectively and quickly that might remove the need for cold stratification. I was also assuming that the gelatinous placental material was very acidic to protect the seeds from contamination from detrimental microbes and the rose achenes would benefit by association and that by providing fresh material the benefit could be extended to reduce the chances of losing rose seeds to contamination as the thickness of the achene was reduced and the first line defence of the achene were breached. What I noticed was that the mixture remained very clean despite being put outside in warm shady place with a muslin cloth over the container. Fungi did grow that resembled penicillium fungi growth that one would see on old orange skins but it looked to be pretty much a monoculture. This formed a skin over the top of the pulp. It gave off a pretty strong odour but didn’t show signs of conventional spoiling.

So yeah… that’s my kind of random thought processes that resulted in me chucking a bunch of rose seeds into mashed up tomato LOL

Hi Simon.

I had never myself seen a bellows-shaped embryo like you showed further up this thread…until today, when I found two twins both flattened into a similar bellows-like shape. Both were gelatinised and looked totally useless to me.

The picture below shows these twins next to their opened achene (their gelatinised texture does not show up well here, as there is too much white glare effect in this webcam shot, which gives a false white color).

What a shame they were duds :frowning:

btw, both had their own individual testa.

The achene came from ‘Flower Carpet-white’ xOP. I have noticed a high rate of embryo abnormalities in the ‘Flower Carpet-white’ achenes (eg. gelatinised or totally dessicated embryos, tetracotyledon, twins).

Update: So far in this test, 50 OP Westerland seeds were planted for each treatment; 50 control, 50 soaked in Tomato pulp for 2 weeks. They were stratified until I saw a germination in one of the bags. The first one to germinate was in the control group. It has, however, been the only one in the control group to germinate. They were sown in a seed tray (normal potting mix) outside in my little greenhouse (in the middle of winter… mild winter so far with min. night temps to -3 degrees Celcius) and the tomato pulp seeds have started to germinate, 3 weeks after sowing. So far am up to 6. Not statitically significant numbers by any stretch but is interesting. The tomato pulp seeds are still germinating at a rate of about 2 a week so far… no signs of any movement in the control group. I’ll have to find my stats book and read up again on how to calculate statistically significant sample sizes so I can repeat it this season in larger numbers. Traditionally I have trouble germinating OP ‘Westerland’ seeds. These seeds were from Don via Paul Barden.

I should also mention that both lots of seed were stratified in bags of moist peat which I was hoping would have additional decomposing processes occuring.

Don,

How are the Mermaid seedlings? Did they survive?

And also…

The ruxburghii seeds I sent you… they were indeed the normalis form, and not the double.

Simon, good show on the Westerland experiment. I lost many of my Westerland OP embryos to contamination so you are doing better than me.

Enrique, sorry to have to tell you that the two Mermaid seedlings didn’t make it. After each sending out two pairs of true leaves they stopped adding new growth. They remained alive for several months until the leaves they did make eventually senesced. It was anguishing to watch, and I maintained the pots for a long time afterward to no avail.

That’s exactly what the OP ‘Prairie Peace’ seedling did for me. Put out first set of true leaves then stopped and eventually died.