A question for all of you out there working with relatively infertile hybrids or ploidy issues. I have worked a little bit with plant tissue culture on magnolia and rhododendron seedlings and was curious about your successes or failures inducing polyploidy via trifluralin. I had experience with Oryzalin and feel that trifluralin would be a good alternative given covid seems to have permanently halted production of Oryzalin based herbicides. Of any successful induced polyploids, have you noticed restored fertility and/or morphologic improvements to your roses? Forgive my naivety with them in particular, I use it mostly for experiments with magnolias to overcome breeding barriers across species. In rhododendron, inducing polyploidy was successful using a suspension of agar and the mitotic inhibitor on newly germinated seed and wonder what your thoughts are!
I love to work with ploidy manipulation too and used trifluralin in the past with rose seedlings. Below is a link to an abstract/paper where I compared trifluralin with cholchicine. Iāve used oryzalin in recent years successfully too. They seemed comparable for rates of doubling for roses. I like to treat young seedlings with droplets suspended between the opening cotyledons. Stressing out the radicles doesnāt make sense since roses are relatively large. Later Iāll isolate and take cuttings off of polyploid-looking stems to overcome what appears to be chimeras at times. I suspect it is more challenging with smaller seedlings like rhododendrons to isolate application of the chemical to just the shoot apical meristem and whole seedling treatment makes more sense.
I have generally done polyploidization to help bridge ploidy levels with plants that still had a bit of fertility at both ploidy levels. One thing Iām aiming to try is to double āCaldwell Pinkā, a sterile diploid with rose rosette tolerance, to see if at the 4x level there could be at least some fertility. I need to get my plant out of the cooler and try treating some axillary buds. I can try to get it in tissue culture too and see if it is better to treat it that way.
Thank you for the interesting information on oryzalin being limited because of manufacturing constraints during the pandemic.
One can buy trifluralin (I suspect in a formulation kind of like treflan is what is in the bottle) for our lawns. I got a small sample of straight trifluralin straight from the company years back and it was very hard to solubilize. Treflan is a formulation that makes a suspension in water (cloudy mix) and at least disperses better than the straight active ingredient in water.
Itāll be fun to learn more about the roses you are working to double.
Thank you David! I appreciate your thoughtful and in depth response! I appreciate the research youāve done on it, I will have to delve into that when I have a moment to sit down. I had to pay for the version sent in springer link but sent a request in ResearchGate to gain access. Did you feel one was easier and/or safer to use for this? My understanding is that colchicine tends to pose more of a toxicity risk to plants and people. Did you find you needed successive treatments to ensure the mitotic inhibitors were penetrating all 3 histogenic layers? Or is it easy enough in roses to just tissue culture the meristem to prevent reversion to lower ploidy? In some of the rhododendron seedlings I worked with, I suspect that I should have used something like DMSO to help penetration to increase efficacy.
I have no doubts that great strides will be made to overcome issues with RRD - its a very unforgiving disease and am curious to see how much we can learn from it. I will keep you updated as I learn more through my trial and error - at the moment I still struggle with damping off or other rot disease of my seeds while stratifying and it seems a perennial issue. These are the roses I have easy access to:
Of particular interest to me would be Polestar - given its hardiness but uneasy ability to set seeds, I canāt help but wonder if this may be one tool we can use to push nature along a bit. Some of the others are very small roses i just purchased and think it will be a while before I have stock of decent size to really make a game plan.
The Generous Gardener
Lady of Shalott
Carding Mill
Boscobel
Claire Austin
Alba Suaveolens
Rosa Virginiana
Morden Snowbeauty
Polestar
Morden Blush
Duchesse de Montebello
Alchymist
April Moon
Cherry Frost
Above and Beyond
Nitty Gritty White
Iceberg
Commonwealth Glory
New Zealand
This is the research that I used as template for my rhododendron seedlings - they take a very unique approach and I love the process.
I loved the article you linked to and thought it was interesting they used agar. I wonder how roses would react to their cotyledons being covered with reduced air exchange long term. I do use DMSO for better penetration and I suspect that helps a lot. Using very young seedlings seems to be key to better doubling before there is much leaf primordia that builds over the growing point making it harder for the chemical to soak in. It seems like the dinitroanaline herbicides (trifluralin and oryzalin) are much more plant microtubule specific and can be used then at much lower concentrations than colchicine to be effective on plants. Colchicine is much more animal spindle fiber specific from my readings, so toxicity to us is a stronger concern for colchicine.
Hereās a link to a video that should be active for a few weeks highlighting some treated polyantha seedlings that are maturing into nice plants. I posted it awhile back, but for some reason the icloud links seem to only last so long and then expire. iCloud
I love your goal of doubling Polestar/Polstjarnan. That would be wonderful!!! I love its hardiness.
Dr. Leo Dionne many years ago developed the āDionneā method for doubling in potatoes and later in his life worked with roses. He was part of the RHA and wrote a few articles for the newsletter. Iām thinking of using his tips for āCaldwell Pinkā. The challenge for a cultivar versus a seedling is that there is a lot of leaf primordia over a growing point and it is hard to get chemicals to penetrate down to it. He would take a razor blade and cut into the bud some to get the chemical through the primordia. He would take a cotton ball soaked in colchicine and DMSO and tie it to the stem over the treated axillary bud for a day or two with a bag over the plant to keep the humidity high and the cotton ball wet. I suspect the agar approach would be even better to hold the chemical in contact and to get it to penetrate over time.
In tissue culture people have tried different techniques. One that seemed to work a bit better was to cut a stem just under an axillary bud and let the chemical soak up the stem to the bud through the vascular tissue coming to the bud from behind versus through the leaf primordia.
I tried multiple treatments in the past, but have stopped at just one moving forward. With DMSO and a concentration of chemical in the target range, it seemed to just kill more seedlings than anything. Maybe since I use a dropper to get the chemical right where it needs to go on seedlings when they are the perfect age, that helped versus spraying over variably staged seedlings where more sprays over time could be helpful.
David, I always check your posts because they are so informative. I have a batch of doublings of my own to attempt this spring. Could you please tell us what specific formula you have settled on that it is best applied to the apical meristem using a dropper?
The following is put into tube and then brought up to 50ml with distilled water.
50ul Treflan (which is 43% trifluralin),
drop tween or soap
and 0.4ml DMSO
It becomes very orange in color. If you store it after it is mixed, it tends to settle and need really good shaking. I like to add water just before I use it and use it all up if I can without storing it.
The techniques i read about seem to typically involve new seedlings and hence generally unknown plants with no established record.
Iām wondering if there isnāt any procedure for e.g. treating axilary buds of very young stems and then removing the apical tip to force development of a newly treated future branch of a known cutivar instead. (If one could treat the node before the leaf was even developed on a rapidly growing shoot is there a possibility the treatment could penetrate?)
Ignorant questions from someone with no formal education in botany, so forgive me. It just seems it would be better to double genes from a known phenotypeā¦
I was wondering if you could also triple a diploid? Or could you treat pentaploids (Canina) and get weird ploidies that would match in a different way to other ploidies? Could you double a tetraploid?
A second question Iām not entirely sure Iāve understood the part that refers to the research of Dr. Dionne. Can you treat buds on a mature rose and hope to, for instance, occulate them on rootstock to get this rose with a doubled ploidy? Has someone done it in the past with roses?
Iām not entirely sure Iāve understood the part that refers to the research of Dr. Dionne. Can you treat buds on a mature rose and hope to, for instance, occulate them on rootstock to get this rose with a doubled ploidy? Has someone done it in the past with roses?