I love Joe’s video tours of his seedlings. He inspired me to record a short video on some polyantha roses I’m working on characterizing for polyploidy. Hopefully you find it interesting. As I was having fun looking at them I had my phone with me and thought it would be fun to share what I was looking at and thinking about. My hope is to try to more easily bridge polys with modern roses. I tried in the past with limited success with more multiflora background polys. These that I treated last year in the video are seedlings that also have a good dose of R. wichurana in them now too. From my limited experience with more wichurana based polys, they seem to bridge and cross more a bit freely (like ‘The Fairy’) and maybe these tetraploids of wichurana based polys will bridge even better. It’ll be fun to find out. Also there are some plants that seem to have 2x and 4x sections and it would be fun to root cuttings and isolate plants of different ploidy levels of the same genotype for additional research in the future (e.g. seeing if there is a ploidy level that is more susceptible or resistant to black spot of the same background like what Andy Roberts in the UK learned with rugosas, etc.).
I attempted at-home, non-laboratory doubling of some practice seedlings using the herbicide Weed Impede as an oryzalin source. I created a 5 uM solution from the herbicide and applied it to the newly opened cotyledons for 12 hours.
For a 1 pint container of Weed Impede with 0.5 lb active ingredient orzyalin:
0.4 ml Weed Impede into 1 L distilled water to yield Dilution 1.
9 ml of Dilution 1 into 1 L distilled water to create second and final dilution, which is the 5 uM solution.
These calculations will be inaccurate if your oryzalin source has a different stock concentration.
I used a graduated cylinder and a handheld engraver to graduate empty liquor bottles. Not the most accurate, but works for at-home use.
References:
“Chromosome doubling in a Rosa rugosa Thunb. hybrid by exposure of in vitro nodes to oryzalin: the effects of node length, oryzalin concentration and exposure time”
“Oryzalin-induced chromosome doubling in Rosa and its effect on plant morphology and pollen viability”
Cool! If you need/want 86-3 or any OP seeds of it, please let me know. I’ve also raised several seedlings using its pollen. All are on HMF. There are two L56-1 X 86-3. One is significantly larger plant with much larger flowers. The other is smaller in all parts, but still a largish climber. The only “repeater” is the Lynnie cross, which I no longer have.
Very interesting!
I haven’t worked with polys yet. Could you explain some of the difficulty faced in bridging with moderns? Is it similar to sterility issues when crossing moderns with Rugosa? Or do they simply not combine well genetically?
Duane
Thanks Jonathan for sharing your well described technique. Duane, I’ve struggled a lot getting seed set between the multiflora based polys crossed with modern tetraploid shrub roses. They do cross pretty well with Ralph Moore’s tetraploid minis though (mainly with his more fertile minis as moms). I have just one successful hybrid from years ago that was generated between a multiflora based polyantha mom (diploid) x ‘Champlain’. I’m excited to try these more wichurana based tetraploid polys with modern 4x shrub roses as moms and see what happens hopefully next year. My hope is to root branches from the plants with polyploid characteristics soon and grow them on in the greenhouse this winter where I can count their chromosomes, look at pollen under the microscope, and collect and freeze pollen from those that seem like they may be more fertile. I’ve used the multiflora based polys with rugosas and got lots of seed set with rugosas as moms, but the seedlings tended to grow weakly and die young. There is one that I saved that has a little fertility and then a seedling of it that has a bit warmer color, so I haven’t given up. Growing op polyantha seed from plants growing near rugosas (mainly ‘Henry Hudson’) I’ve found some seedlings that seem to be natural hybrids too. I’ve only used some of the diploid wichurana influenced polys in a limited manner, but was impressed to actually get some Carefree Beauty x Pink Gnome seedlings years back when I couldn’t get any seedlings with CB or other easy females with the straight diploid (or even tetraploid) multiflora based polys I mainly had at the time. I’m excited for what may be possible with these new 4x wichurana based polys.
I like the idea of making your own graduated measuring devices. I found that olive oil bottles make perfect 250 mL graduated bottles. And with a kitchen balance, I don’t even need to bring home a graduated cylinder from work or buy one. I weighed out masses of 50, 100, 150,200,250 g on the tare wt of a well cleaned olive oil bottle. I simply used a nursery marker for miniscus marks.
More recently I found that a bottle of graded Kraft or Aldi parmesan cheese has ridges naturally placed correctly. So no marking need if plastic is OK for your work.
David, thanks for this interesting video and your explanation of what to look for as key characteristics of a plant with doubled chromosomes.
Your efforts have used fresh seedlings. Do you have any advice for technique when trying to double the apical meristems of more mature woody plants like fruit trees? Would timing make a difference with respect to the best season? Would you need more or less concentrated solution of oryzalin, or perhaps the use of DMSO?
That’s a great question. It seems harder with all the leaf primordia over the growing point. Dr, Leo Dionne developed a technique named after him with potatoes years back (1960’s I think). He was interested in roses in his later years and was part of RHA for a bit. He would advocate for taking a razor and cutting into an axillary bud so the chemical soaked in, using a saturated cotton swab (with chemicals) for a day or so wrapped around the stem, and then wait to see what grew out. The main growing point was typically killed of the bud, but some to the side buds to the main bud would develop some. There’d be multiple shoots that come out and he would screen those. Years back I tried that with some polys and trifluralin (with DMSO and a drop of soap, etc.). I got stressed buds and those that grew often had multiple shoots. It seemed like only part of some leaves were doubled that expanded and it was hard to find a stable total 4x shoot. Maybe it was hard to get the chemical soaking in as far as needed? Compared to seedlings, there was a lot of power to force out the shoots, which is nice. I’m not sure about increasing DMSO. It really stresses and damages tissues. I think the key could be to focus on getting the chemical into the tissue itself. Maybe a syringe would be better than lots of scraping and cutting with a razor, or combo of both? I like the idea of a moist cotton ball to keep the stressed and wounded tissue from drying out at first so the chemical can soak in and be effective. I’d really love to double ‘Caldwell Pink’ to try to see if it restores fertility and maybe the partial rose rosette resistance it has can be transferred to the next generation. I suspect for a season, a time of the year with moderate growth and moderate to cooler temps may be best (spring or fall). Active growth is important to have cells in mitosis to interfere with and then at cooler temperatures it slows things down a little bit and lengthens the cell cycle for division giving more time for the chemical to act and interfere with spindle fibers.
Slight tangent, but did the more multiflora polys that didn’t cross well with moderns, stem from the angel rose/wings seed? I know you had selections of those in the past. Probably doesn’t help at all and who knows how distant from each other the seed strains are at this point, but may be of interest to someone…they accept r. glauca pollen resulting in seedlings with the same cold greyish top and red under side foliage. Granted no idea if that would assist at all in bridging or fertility at all at this point.
for colour comparison
top left - derived from angel rose/wings seed, top right - some (runty) things from sweet spot calypso op
Just searching for the most recent article on Oryzalin to ask a follow up…
Apparently Surflan has been discontinued, and I wondered if folks had a new affordable source for Oryzalin. I keep fantasizing about doing experiments, first with some of my less valuable OP’s…
You can get trifluralin through this Monteray product with the link below. It is basically liquid Preen and is the product often called by the trade name Treflan. From my understanding, Preen is just ground up corn cobs as a carrier with Treflan sprayed over it.
Great question about availability. Most researchers use oryzalin. In the late 1990’s I came across an article by Japanese researchers working with corn and their use of trifluralin. Treflan was available to me from the weed scientists on campus. For some reason oryzalin just became more popular for use in doubling the past couple decades. I got some Surflan a couple years ago or so an tried it and I can’t say there was a clear indication it is better or worse than Treflan. The treated polyantha plants are building nicely and in a few weeks should start blooming for the season. I should record and post another video of them at this point. It is fun to see the leaf differences.
One of the studies I have read stated this about that topic:
“Oryzalin may be regarded as a preferred chromosome-
doubling agent because it is safer for researchers to
use than colchicine. Additionally, colchicine requires a 1000-
fold higher concentration to produce quantitatively similar
effects (Upadhyaya and Nood´en, 1977).”
And, from what I gather from reading studies, it (oryzalin) seems to more frequently produce a wider variation of rare ploidy states. I say more frequently, yet with either chemicals, the odds are still low.
While Surflan is more of an estrogenic that a carcinogen, I still use heavy gloves and only use it outside. I don’t want to find out it has a side effect yet not known.
Good point why dinitroanaline herbicides have become more popular than colchicine for chromosome doubling. Both orzalin and trifluralin are dinitroanaline herbicides (used as preemergents) and are similar in how they work. They supposedly target slightly different parts of microtubules of spindle fibers during cell division to disrupt the process and arrest cells part way through mitosis. Animal microtubules supposedly are much more sensitive to colchicine than plant microtubules and visa versa for the dinotroanaline herbicides. So, we can use a much lower dose/concentration of the dinitroanalines to get the impact we desire in our plants than the dose needed of colchicine, like your reference stated. Thankfully the combo of having a much smaller dose of dinitroanaline herbicides AND that they are less toxic to humans have contributed to the decreased use of colchicine for chromosome doubling. I always wear gloves and my respirator just to be extra safe, especially since I also use DMSO in the mix. DMSO helps to make cell walls more permeable to allow whatever other chemicals in the solution to soak in better.
David, thank you for this video and especially for illustrating how the three layers of tissue in the seedling are affected differently by chromosome doubling techniques. Maybe at some point you would also make an instructional video on counting chromosomes for us mere mortals. I’ve recently resurrected an old microscope that Henry Kuska gave me with an eye to doing just that.
