Chromosome Doubling Experiment

I’ve been having fun treating seedlings with a trifluralin solution in an attempt to double the chromosomes.

Here are some pictures, which are probably in reverse order of what I intended.

Open-pollinated seed of Lena.

Thanks so much to my friend for providing Trifluralin and DMSO in a little vial to which I needed to add 50 ml water and a drop of dishsoap to make the proper dilution. Instead of water and soap I used Freez-Pruf, a product that contains polyethylene glycol and probably some good surfactants. It ended up looking just like orange juice.

A little drop between the cotyledons as soon as they opened. I treated seedlings as they popped up for about three days, then stopped. The untreated seedlings in the first picture are actually younger than the treated seedlings. The treated seedlings look quite stunted, but had good roots. I take that as a good sign that the chemical was doing its job. So maybe there is a chance that some of these seedlings that look like they’re barely able to push out new growth have been doubled.

Overall I transplanted 141 treated seedlings.
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Good luck! You’ll have to keep us posted on the progress of the treated seedlings.

Quite a few of the seedlings are starting to push out some sort of growth. In many cases the new growth is distinctly lighter colored than the first leaflets. It almost looks like an aphid nestled down in there. (And I have a few aphids around, so sometimes I have to touch it to make sure.) A good sign, I wonder?
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They look promising Joe!!! It will take some time to see how the shoots stabilize and which cells overtake the meristem. The growth that emerges from them eventually for color, dimensions of length and width of leaflets, etc. will be very telling for which are the promising ones. Eventually looking at stomate size and pollen diameter under the microscope will help confirm the promising ones for doubling in layer I and II of the meristem.

I just did a conversion of about 100 seedlings of Prunus tomentosa (Nanking cherry). I am interested what concentrations of each chemicals you used?

Hi Johannes,

That is super cool! I hope you will report on the results. Did you do the same method of putting a drop of treatment between the cotyledons?

David Z was my angel for this project, and I’m assuming the percentages are the same as he described in his paper
“Trifluralin-mediated polyploidization of Rosa chinensis minima.” That is, 0.086% trifluralin with 1 or 2 percent DMSO. They were dissolved into a product called FreezPruf.

What chemicals and concentrations did you use for your cherry project?

I used the same concentrations but always at 1% DMSO. I treated the plants with 5ul of solution the petioles started to split near the cotyledon. I find that one treatment is usually enough. I estimate about 50% conversion. It is so much easier than the old colchicine. I used to get only 1% conversion and toxicity problems not to mention toxicity to the user. It is also too expensive.
I got this info from Euphytica vil 141, Issue3, pp 281-290

Joe I do not want to sound too critical, but I would like to see a bit more disortion in the new growth to be absolutely positive something had changed. I use concentrations of 1: 80,000 Trifluralin, applied the same way as you but kept in a high humidity chamber for 24 hrs. The distortion I am talking about should look like this.

Cheers Warren.
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Hi Warren. I, too, kept them wrapped up to give high humidity for 24 hours.

An update: many have died. I don’t know if it was from the treatment or simple damping off.

There are many that were quite stunted, similar to those in your pic. They are now mostly in the next stage of trying to push out some growth from below. There are some that have pushed out a lot of new growth, and appear to be non-conversions. I am most hopeful for those that are just now barely able to push out some new growth.

Nothing to do but wait and see! It’s fun to have something to look at during the winter.

Warren, do you have any pics of those seedlings at a slightly later stage when they were pushing out some new growth?

Joe the new foliage when it comes through will look normal, it takes a while as you can imagine what it would be like having your insides turned upside down. I lost a couple from damping off but most where discarded due to PM problems. From the literature I read, it is most effective on diploid types, and to a certain degree triploids. Tetraploids are not used. This the pic of the Mutabilis X Mutabilis treated with Trifluralin, the foliage is still very China like and has continuous flowering for 9 mths untill I give it a trim in winter. It looks like this every day , always with bloom.
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That’s a lovely one, Warren.

Warren, do you have any photos of your mutabilis to compare with this charmer?

Don that is the beauty of knowing people with huge rose gardens, you do not have to purchase huge numbers of roses to breed with. The mutabilis used was in a friends garden and just looked like any other mutabilis, nothing special, but as a lot of the old type roses, records of parents were not recorded untill modern times. It makes you wonder what Mutabilis really has in it.

It makes you wonder what Mutabilis really has in it.

Yes indeed. I grew some OP Mutabilis seedlings that were very nearly vines but they couldn’t take the winter here.

just looked like any other mutabilis

I got a close look at a fairly large Mutabilis growing on the perimeter fence at the UC Davis student-run Independent Garden Center facility, so I’ve seen what it can do in it’s preferred climate. Your seedling has far more flowers on it and the pigmentation seems to be more dense. Now, if you could inject some cold hardiness into it…

cross it with R. virginiana, somehow recover repeat…

Back twenty-five plus years ago, I raised a Mutabilis self which was white with spring only flowering. It was VERY vigorous and rather rampant. I culled it as it was nothing I hoped for at the time and room and water, though much more plentiful than now, weren’t sufficient to retain something of that nature.

Joe this Mutabilis seedling was used a pollinator on a hybrid Kordesii and has formed a huge hip. This should inject some cold hardiness into the genetic pool.

Warren

I completed the crosses with measuring stoma size. It proved harder that I thought. 6 plants were indicated and two of these were obviously. I will inter cross these hybrids to hopefully produce a stable race of tetraploids. johannes

Isn’t that how it is, Johannnes? No matter how many seedlings you grow, you end up with just one or two winners. For those of you just seeing this thread, he is working with Prunus tomentosa. It will be fun for you when they reach bearing age!

Since you brought this thread back to the top, I should report on my treated seedlings.

My control group is made up of seedlings that germinated slightly later than the ones I treated; I’d say by about a week. Now, I suppose that technically could be another variable. Since I was dealing with OP seed, maybe the seeds resultant from outcrosses with tetraploids could have been prone to germinate slightly earlier??

Barring such a phenomenon, there are some clear morphological differences between some of the treated seedlings and the control group. Several (or maybe closer to “a few” or “a couple” of them) have clearly wider leaflets than any of the ~80 seedlings in the control group. I tried looking at pollen diameter with my little pocket microscope, and it seemed to me that at least one of the treated seedlings had larger pollen than at least one of the untreated seedlings. However, not enough blossoms were happening at the same time to get an accurate sample.

Somebody to whom I was showing my experiment noted, without any knowledge of that characteristic being a possible result of polyploidy, that two of the treated seedlings seemed to have more petals than those in the control group.

it will be fun to continue to observe these seedlings as they grow and to get more pollen samples. Maybe even send some pollen to someone with a better microscope. I will post pics for y’all sooner or later.

In accordance with the rule I mentioned at the beginning of this post, I expect to end up with one or two keeper plants that have the potential of being tetraploids.

This year I applied a Trifluralin solution to these Rugosa seedlings when at the cotyledon leaf stage. After 5 months this is how they are going. The first pic the seedling was planted out in the growing beds one month before the others, it now is about to flower. The other two do show some slight difference in foliage type. The seedling about to flower is no more than 10 inches high.


Warren
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