I was doing some more cytology this weekend and was surprised to learn that the floribunda ‘Nearly Wild’ is a triploid (21 chromosomes).
The fertility seemed kind of low to me as a female and I never tried it as a male. I just got a handful of seedlings from it over the years and haven’t saved any. What have others experiences been with using it as a parent?
Sincerely,
David
Dave I have had a few open pollinated seedlings from Nearly Wild see: http://home.neo.rr.com/kuska/nearlywildseedlings.htm
I do not remember using them in further generations so I cannot tell anything about their breeding behavior.
Hey David,
I’m glad someone out there is trying to make some sense of all these roses whose ploidy we still don’t know. Any other roses you’ve found the ploidy on?
Mike
Hi Mike!
Yes, in the next newsletter there’s an article on the technique I’ve been using and some more roses that have been confirmed. Here’s some of those and more recent confirmations:
Tetraploid
John Cabot
Carefree Beauty
Summerwind (Buck one)
Heritage
Carefree Sunshine
Dorcas
De Montraville
Sir Clough
Diploid
Lillian Gibson
Sincerely,
David
Thanks David,
I notice you have confirmed Heritage and Sir Clough - two English Roses. However, I am fairly certain that most of the English Roses are tetraploid, just by looking at their backgrounds - since the English Roses are heavily inbred, it is fairly safe to assume that the great majority are tetraploid.
Mike
Good point Mike.
I’ve been practicing the technique on plants I just have in the greenhouse and have tissue available of now. In the future if people have roses they would really like to know the ploidy of, I’m willing try confirming them as my time and availability to plant material permits.
David
David, would you one day publish your technique on the way of doing this in a home setting? I’m anxious of seeing if I’ve induced in a Roseraie de l’H
Enrique, I found an interesting page covering a technique for ascertaining chromosome numbers that you might be interested in glancing at. It’s http://www.bridgewater.edu/~lhill/chromtechnique.htm[/url]. I don’t know how easy or practical it is to set up and do at home, but I had been wondering about the process myself as I was curious about ploidy in some plants I have. Anyway, happy reading!
I wonder if Preem could be substituted fot the colchicine in the following method:
Title: A New Procedure to Prepare Slides of Metaphase Chromosomes of Roses
Authors: Yan Ma, M. Nurul Islam-Faridi, Charles F. Crane, David M. Stelly, H. James Price, and David H. Byrne
http://www.electronicipc.com/journalez/detail.cfm?code=04200020310532
ABSTRACT. “To our knowledge, there has been no published technique to produce consistently high-quality slides of somatic chromosomes of roses (Rosa sp.). Therefore, various pretreatments, fixatives, digestions, stains, and maceration and squashing methods were tested to identify a procedure to produce clear, well-spread chromosomes from shoot tips. The best results were obtained after pretreatment in a mixture of 0.1% colchicine and 0.001 M 8-hydroxyquinoline for 4 h, and fixation in 2 acetone : 1 acetic acid (v/v) with 2% (w/v) polyvinylpyrrolidone. The darkest-stained chromosomes were obtained with carbolfuchsin staining of air-dried cell suspensions that had been spread in 3 ethanol : 1 acetic acid (v/v).”
Link: www.electronicipc.com/journalez/detail.cfm?code=04200020310532
David, if you haven’t done it can you do ‘Will Alderman’? It would help determine if Rosa acicularis or woodsii is in its pedigree. Thanks.
Paul
From the reading that I have done, I get the impression that almost as important (if not as important) to the actual "X"aploid of a rose is what distribution of “plodies” it produces. It appears that one can get an idea of this (for the pollen) by just looking at the size distribution with a “toy” microscope. I will try to find some of the references for this when I have time. Will Alderman is one that I suspect produces a mixture of various chromosome number pollens.
Another point, I recently read that if one finds 2 seedlings in one seed, most often the smaller is not one with half the chromosome number ( as had been thought) but one that is just 1 or a few chromosomes short of normal. This is why the second seedling often dies in spite of out attempts to keep it alive. I spent some time looking for where I filed that article, but so far have not found it.
This is one of the papers about the distribution of ploidy pollen.
Title: Evidence for the production of unreduced gametes by tetraploid Rosa hybrida L.
Published in: Euphytica, volumn 133, pages 65-69, (2003).
Authors: L. Crespel (1), S. Gudin (2)
Authors affiliation: (l)Meilland Star Rose, Domain Saint Andre, 83340 Le Cannet des Maures, France; (2)Universite d’Aix-Marseille III, Faculte de Saint Jerome, Service 442, Av. Escadrille Normandie-Niemen, 13397 Marseille Cedex 20, France.
Abstract: “Dihaploid roses, parthenogenetically derived from Rosa hybrida L. tetraploid cultivars, were recently shown to produce unreduced male and female gametes. On the occasion of an attempt to obtain new dihaploids, the gametic behaviour of some tetraploid R. hybrida cultivars used in the haploidization program was studied, as some deriving varieties unexpectidely revealed tetraploid. The pollen grain sizes of the original tetraploid cultivars were measured and a cytological analysis of the male meiosis was carried out. And AFLP analysis of these cultivars and their derived tetraploid varieties was made. It was used to determine the genomic contribution of the original cultivars in their derived forms. The results obtained tend to demonstrate that unreduced gametes can be produced by tetraploid roses. Perspectives for the use of such 4 x gamete producing roses are discussed, as well as the adaptative value of such high leveled ploidy gametes.”
Paul, if you can send me a plant of ‘Will Alderman’ or prepare root tips for me and send them, I’ll try to confirm it. I don’t have one and the rose labeled as WA at the MN landscape arboretum is actually ‘Therese Bugnet’.
Henry, good thought about trifluralin to arrest cells at metaphase. That would be a great experiment for someone to optimize its use in this regard. Instead of using colchicine like Ma et al. or trifluralin (I tried it) these days, I just use ice water for 24 hours. It has been good enough and I don’t need to worry about “proper” disposal of these chemicals.
In the article I wrote a few years ago on haploid extraction I cite some references describing twin embryos. The second embryo can be a synergid that developed into a second embryo and would have the same chromosome constitution as the unfertilized egg and be haploid. In corn that happens in ~1 out of 700 seeds and in flax researchers have done recurrent selection to develop lines that produce a second, haploid embryo more routinely. The trick is to have parents with little genetic load to be able to recover many of these secondary embryos and have them grow on to mature plants.
Since chromosomes are very small for roses, without a 10x eyepiece and a 40x objective with a good light microscope, it would be hard to do the technique. Beyond the microscope and some slides and cover slips and a razor blade there isn’t too much else one would need. One would just need some acid to soften the cells (maybe those enzymes you use for seed germination Henry would work) and some acetocarmine for stain. With good eyes and a phase contrast objective, the stain isn’t really necessary.
Sincerely,
David
I found the 2 eggs in one seed article:
Title: Sexual polyembryony in almond.
Authors: Martinez-Gomez, P.; Gradziel, T. M.
Authors affiliation: Department of Pomology, University of California, One Shields Avenue, Davis, CA, 95616, USA.
Published in: Sexual Plant Reproduction 16(3) September 2003. 135-139.
Abstract: " Multiple embryos within the same seedcoat occur spontaneously in certain almond (P. dulcis (Mill.) D.A. Webb) cultivars including ‘Nonpareil’ and ‘Mission’. Seedlings from the same polyembryonic seed are frequently viable, though one of the seedlings often shows weak growth and develops poorly. These dwarf seedlings have previously been reported as haploid. In this work, we have characterized several seedlings from ‘Nonpareil’ polyembryonic seed, including their germination and later growth. Isozyme and simple sequence repeat markers were used to analyze seedling genetic structure. In addition, individual mitotic karyotypes were determined following root-tip staining. The percentage of twin embryos showing aberrant growth was approximately 30%, with mortality rates of about 90%. The majority of these aberrant seedlings appear to be aneuploids. Most secondary embryos appear to be derived from the primary embryo following normal fertilization."
I am pretty sure I also have the full paper. The question is where.
While looking for the paper mentioned in my post above, I found another full paper that I have.
Title: Ploidy reduction in blackberry
Authors: Naess, S. Kristine; Swartz, Harry Jan; Bauchan, Gary R.
Authors affiliation: Address Dep. Horticulture, Univ. Maryland, College Park, MD 20742, USA.
Published in: Euphytica, volumn 99, pages 57-72, (1998).
Abstract: “Polyptoidy in blackberries and ploidy differences between Rubus species are obstacles to the efficient introduction of valuable germplasm, both intra- and interspecific, into blackberry breeding programs. Expansion of the germplasm base would be facilitated by reducing the ploidy level of blackberry cultivars to the diploid level. In this report selection of twin seed, interspecific hybridization, and pollen irradiation were compared as methods in the recovery of dihaploids from tetraploid blackberry cultivars. One dihaptoid was obtained through selection of twin seed and several were obtained following interspecific hybridization. The infrequency of twinning and difficulty in detecting twin seed in Rubus reduced the efficiency of this method. The efficiency with which dihaptoids could be obtained following interspecific hybridization varied with the pollen parent. Reduced seed set and seed quality following pollinations with respectively R. parvifotius and R. hirsutus could be used to advantage in the recovery of dihaploids from blackberries. Ploidy reduction in several tetraptoid blackberry cultivars was obtained following pollinations with 100 and 150 kR gamma irradiated pollen. Most of the seedlings obtained at the 50 kR dosage were aneuploid. Pollen irradiation at 150 kR was the most efficient method of obtaining dihaptoids from tetraptoid blackberries. Twenty percent of the seedlings obtained following this treatment were dihaploid.”
One page 68 they state: “In the course of pollen treatment and interspecific hybridization experiments conducted with blackberries (Naess, 1995) 14,371 seeds were dissected. Five sets of twin embryos were found and four sets germinated and survived. One set consisted of a diploid-tetraploid pair and the remaining three pairs of seedlings were mixoploid with chromosome numbers ranging from 15 to 42.”
I found the full “Sexual polyembryony in almond” paper that I referred to earlier in this thread.
The introduction gives what looks like an excellant review of previous “twins” research.
In the Results and Discussion section, the following appear:
"Isozyme variation
A comparison of AAT and PGM isozyme patterns showed similar polymorphism for embryos from the same seed (Table 1). These results support a similar origin for both embryos in twins regardless of dwarf or normal phenotype.
Different alleles were present in different polyembryonic seed for AAT-1/AAT-2 and PGM-1/PGM-2 isozymes. These results do not support a haploid genotype as proposed by Gulcan (1975) for dwarf almond seedlings. In haploid peach selections, Arulsekar and Parfitt (1986) reported the absence of malate dehydrogenase isozymes, as would be expected in true haploids."
And:
"SSR variation
Most SSR markers also supported an identical origin for embryos from the same polyembryonic seed regardless of dwarf or normal phenotype. However, certain markers were absent in some dwarf seedlings (Table 1; Fig. 2). Lebegue (1952) had previously reported finding genetically similar sexual polyembryos in wild strawberry (Fragaria vesca). In citrus, Cameron and Garber (1968) reported that genetically identical polyembryos might occur following interspecific hybridization.
The absence of certain SSR markers in some dwarf seedlings may indicate the loss of a chromosome resulting in monosomic aneuploidy rather than haploidy. Diploid-aneuploid twins, however, have not previously been reported. Maheswari and Sachar (1963) reported that the most common situation for polyembryonic embryos in plants is a diploid-diploid or haploid-diploid combination. Kimber and Riley (1963) also proposed that multiple embryos are an important source of haploids in angiosperm plants. However, Specht et al. (2001) described an uneven distribution of chromosomes in some plants from polyembryonic seeds in species of the genus Allium. Allard (1960) describes a similar dwarf growth phenotype for monosomic aneuploids in several species. Swanson et al. (1981) concluded that the loss of a chromosome in a diploid organism is even more deleterious than haploidy or the addition of an extra chromosome because the chromosome balance is disturbed and deleterious recessive genes are expressed."
"Karyotypes of root tips from polyembryonic seedlings showing normal growth had a diploid chromosome complement (2n=16), while an aneuploid chromosome complement (2n
Like Henry said, pollen diameter is a good way to estimate ploidy. I use that method often. Sometimes with lots of aborted pollen or 2n pollen sometimes it can be questionable what the ploidy is of the parent we want to use. Unless you have access to those expensive slides (micronomers?) to calibrate the hash marks one may have in the eyepiece of their scope, having a range of diploid and tetraploid pollen diameters in uM will do little good. One has no way to measure in uM. I suggest looking at pollen from known diploids and known tetraploids and then compare the size of your pollen. Generally pollen from tetraploids have pollen that is 1.2-1.4 times the diameter of diploid pollen.
David
I have gotten the impression that there are inexpensive “toy” microscopes that feature computer outputs. Does anyone have any experience with one of these? This question is not for my own use as I have been able to purchase a very good quality used microscope from a store that sells to homeschoolers.
I have one of those things Henry. It won’t work with my computer. You can have it if you like if you help me out with the shipping costs. It seems to work, and doesn’t seem to be broken. My computer has so much problems for me, and so it’s been collecting dust. I bought it for 40 dollars on Ebay, which used to be sold for 150 dollars when it first came out in 1999 or around that time.