Thanks for the abstract on the begonia article Henry. From my potato work the key was to have homozygous recessive genotypes for the genes governing parallel spindles or some of the other meiotic mutants. There is literature in the potato world too that after that point, one can do recurrent selection within homozygous recessive material for increased rates of 2n gamete expression. In my chromosome doubling rose paper I identified a diploid polyantha that produced 2n pollen (selection 4BA3). Later I used it as a parent with ‘Orange Honey’, a tetraploid, and had a nice orange blend triploid seedling. I raised op seed of it (it was surrounded by diploid polys) and got a diploid seedling from it (1W13) and for a subsequent paper on pollen diameter and ploidy learned that it also produced 2n pollen.
I’m excited about the different ways (nitric oxide, etc.) people are finding that they can treat plants with to induce 2n pollen even in genotypes that normally don’t produce them. I think you posted some of those abstracts in the past Henry for us.
If I didn’t want to double the ploidy of pollen (or ovules) at pollination but instead wanted to try and force chromosome doubling in diploid regenerative tissue to propagated tetraploid versions of the same plant how would you go about it (safely)?
David, I found the paper you mentioned above (see link). Did you succeed in making tetraploid R, chinensis minima that then made 2n pollen?
Link: www.springerlink.com/content/v57055q8535x7527/
Hi Simon,
I was able to generate several 4x polyanthas and still have some growing in my garden. I just happened to find the diploid that wasn’t chromosome doubled that produces 2n pollen. One of the induced 4x polyanthas is the male parent of my Honeybee pictured on the member showcase section of the RHA website.
Sincerely,
David
‘Honeybee’ is lovely. So did you use trifluralin to double the polyantha? I’ve also been reading about something called oryzalin. What do you think about the idea of making a trifluralin solution and standing short (3 or 4 node lengths) laevigata, clinophylla, and gigantea cuttings in it for 24 hours. I also read somewhere that uptake was more effective through the cut ends of short nodal cuttings (for tissue culture) than by absorbption through the tissue. One group even injected it directly into the apical buds. You found 0.086% to be the optimum concentration didn’t you?
“I think it would be great to pollinate GA with a diploid that has very uniform pollen size and a tetraploid male with very uniform pollen size typical for its ploidy level and look at the relative ratios of ploidy levels among the offspring. There is some evidence that there is preferential embryo development (yet not complete) from gametes having the same ploidy level.”
David,
I’m not sure if these crosses qualify, but I have the following: ‘Golden Angel’ X ‘Reve d’Or’, and ‘Golden Angel’ X ‘Champlain’. I have assumed for years that ‘Reve d’Or’ is a diploid, as so many Tea derivatives are, and ‘Champlain’ is a known tetraploid. We’d have to confirm ‘Reve d’Or’, and I know nothing about the uniformity of pollen sizes. Anyway, I have many seedlings from both crosses that can be sacrificed for the sake of experiment. I plan to cull most of the ‘Champlain’ cross soon, so I need to know if you want me to hang on to them.
Paul
This thread has been very interesting reading and very educational. I was wondering how a person could declare a seedling to be a new species and have the rose breeding community accept that at face value, if that is what happened at the time. Was there any scientific questioning of the type that has been posted here? If this had occured in 2010, as opposed to 1950, would Rosa x kordesii be a more appropriate label? Again, great information here. Thank you all.
Rob
Hi Simon,
Yes, it was trifluralin that doubled the seedling. I actually was going to try to chromosome double three different diploid polyantha genotypes I really liked for the experiment. I had lots of cuttings of each genotype stuck to later apply all the treatments. Something happened to the mist system over a weekend my cuttings died. Instead of giving up, I decided to use seed of the clones. I’m SO glad it worked out that way, even though I was very disappointed at first. In my side experiments with treating axillary buds on larger plants, it has been very very difficult. THere is a lot of leaf primordia over the growing point to get the chemical through. For seedlings and using an eye dropper as the cotyledons just crack to hold the drop, there is a lot less material over the growing point and relatively high rates of doubling. I LOVE your idea of using cuttings and making a cut just below a node so the distance the chemical has to move to the bud is short and then rooting it and forcing out that bud. Very clever and a nice application of the tissue culture information from Andy Roberts and his colleagues. I think you may really be onto something! I suspect the treatment will interfere with rooting and lead to some necrosis, but as long as we are patient and have a good setup to keep the cuttings limping along until rooting we should be fine.
There is the Dionne method published in the 1960’s that takes a razor and cuts into an axillary bud and then treats it so there is better penetration. I tried that in potato and rose too. For efficency without tissue culture, I still have a hard time imagining a technique with trifluralin/oryzalin/colchicine that would be more efficient than seedlings. I have a nice discussion about the pros and cons of seedlings versus known clones for doubling in that article.
Take Care,
Sincerely,
David
Ahhhhh… callousing and root formation is mitosis isn’t it… hmmm… maybe a weaker concentration might be enough to do the trick… would the addition of liquid rooting hormones make any difference?
Rob,
I would say it was likely because of Dr. H.D. Wulff’s stature is the reason why he wasn’t challenged more at the time regarding him giving the species name Rosa kordesii. This kind of situation wouldn’t happen today.
Thanks for the reply Paul.
Is it possible to induce something multiple times to ploidy such as hex,oct, etc? It’d be fun to breed modern roses at that level just for the sake of curiousity lol.
Hi Jadae,
Yes, in some of the past polyploidization articles plants that are doubled twice are reported, but I think they generally have been quite weak and slow growing.
Bummer 
I wonder what would happen if any of these were to survive and bred with something of similar ploidy but with a more mixed septet formula?
David, you said that continuous doubling resulted in a lose of vigour. Does doubling chromosomes usually result in a drop in vigour? Should we be worrying about doing this if it’s only going to result in less vigourous plants?
I went and tried to buy some trifluraline the other day from my local ag. supply store. They looked at me as though I was some kind of terrorist… can see I’m going to have trouble sourcing it…
Simon,
Ask for Treflan. I dont know the laws of AUS, so I dont know if it is the equivelent to the US laws of RUP (Restricted Use Product) vs GUP (General Use Product). The former requires liscensure to even purchase it. Anyone can purchase the latter. I, someone that holds an applicators license to apply any RUP, cannot even buy a RUP. I wish I knew more about pesticides in Australia so that I could help ya out.
Hi Simon,
That is a great point and question about ploidy and reduction of vigor. Going up in ploidy is not always the best. My 4x polyanthas have a slower growth rate and less branching than the 2x polys. I even had some adventitious shoots from below the cotyledons of some seedlings to compare 2x / 4x versions of the same rose.
Decades ago as people doubled daylilies to get some 4x ones they supposedly weren’t that great. It wasn’t until people intermated those to increase heterozygosity at the different loci or genes did we get the interesting hybrids of today. Maybe later matings and selections at the 4x level will increase some vigor generally. I think that ploidy is not the only factor of course influencing vigor and we can find exceptions, but generally I think it is true that as we go up in ploidy the growth rate declines- at lease for roses and other plants I’ve worked with. People are in awe of the larger plant parts with polyploids that I think in general discussions this point is sometimes ignored.
Triploidy is super common now among the vigorous landscape roses of today. I think it is common and is a byproduct of general selection (I suspect most breeders aren’t carefully documenting their ploidy) for roses with quicker growth rates, reduced fertility for quicker rebloom, etc. It is a nice balance between the generally thicker petals, etc. with polyploids and branching and quicker growth rates generally associated with lower ploidy levels. Of course we need to keep in mind genetic background and other factors that influence those things, but ploidy definately impacts those traits.
Some roses are better adapted and stabilized at higher ploidy levels (Caninae section, etc.). Some roses it seems in colder or harsher environments have generally higher ploidy levels. Some think it can house more and different alleles to help survive under harsh conditions. In warmer climates, there is a greater ratio of diploid roses where fast growth rates to compete with other vegetation is important perhaps.
I love working with ploidy and enjoy counting chromosomes. Hopefully more of us will be willing to do it. There are other, indirect, things that can be measured that generally point to a ploidy level (pollen diameter, stomate size, etc.) and there are good reasons to measure and work with those pieces of data, but if one wants to know the ploidy level of the original plant, directly counting the chromosomes in the cells is the way to go. Roses do have three distinct layers in the growing point and there is a very small chance they can differ in ploidy, but we can look at that too by assessing chromosome numbers in different types of tissues as well.
Take Care,
David
P.S. The strangest plant for ploidy I found was a 6x seedling from an op seedling of a diploid hybrid of Rosa setigera x a polyantha seedling. It had a very slow growth rate and rounded leaflets, typical of polyploids. It has the floral traits of the Synstylae section and no visual evidence of traits from higher ploidy Caninae section roses I also have in the garden.
So… this might be an ‘out there’ kind of question… can you induce a halving of chromosome number instead of doubling it? Can we make diploids out of tetraploids?
Hi Simon,
Henry’s article is great and his experience generating callus. It is very difficult to regnerate shoots from the callus, like he mentioned. I also tried anther culture too and was able to get more anthers generating callus with silver nitrate in the media to bind up ethylene sites.
I have successfully obtained some haploids over the years through being very observant of seedlings. Sometimes unfertilized eggs can form into embryos. A number of years ago I crossed some tetraploid shrubs and minis with roses I know are 1x bloomers and should not carry alleles for repeat bloom. I expected all one time blooming offspring. Some repeat blooming offspring seemed out of the ordinary and they had more petite leaves and flowers. I counted their chromosomes and some were diploid. Most were relatively weak and have died over the years unfortunately. Keeping our eyes open for roses that have plant parts that are smaller and thinner and more branched habits can guide us to plants we could count and find some that are diploids. In addition, we can go through multiple generations and raise seedlings from triploids and get some diploids as well.
There is a French group that made pollinations of hybrid tea cut roses mainly that irradiated pollen and then did some embryo rescue with tissue culture and that process helped them generate a number of 2x roses from tetraploid parents. A relatively high proportion of eggs were able to develop into embryos without what seemed to be fertilization. The pollination process somehow helped with eggs developing into embryos and then rescuing them and growing them in tissue culture helped them from failing on the parent plant it seems.
In addition, in crosses with tetraploid cultivars or advanced seedling selections I have obtained triploid offspring. I think with such mixed backgrounds and lack of pairing in a normal fashion and some chromosomes being lost along the way, some gametes are produced with less than expected ploidy. For instance, ‘Prairie Harvest’ is triploid and both ‘Carefree Beauty’ and ‘Sunsprite’, its reported parents, are tetraploid.
I suspect a lot of relatively rare 2x plants from a 4x female that may be from an unfertilized egg are discarded as weak. At the 4x level plants can carry along a lot of deleterious alleles. This is often called genetic load. When we go down in chromosome number some of these alleles are no longer masked and gametes if grown into plants themselves may have weaknesses from those alleles that are exposed. It is often manifested in reduced vigor and a high rate of them that are sterile. In my work with potatoes and doing the same thing with them, most of the diploids from tetraploid parents were sterile too.
Some roses have less genetic load I think and I have recovered more 2x plants from them from unfertilized eggs than other roses that have better than average vigor. 'Rise ‘N Shine’ and ‘Dorcas’ have each produced 2 or more diploids over the years that I was able to confirm.
It is amazing how in nature there are mechanisms in place to go up and down in ploidy.
Sincerely,
David