Doubled Haploid Technique explained - video

This is a really good explanation of what dihaploids are, why they are useful and how to make them (with maize).

Dihaploids can happen in roses and it is my belief that this is what happened when Tantau crossed roxburghii (which would be the ‘inducer’) with Baby Chateau to produce Käthe Duvigneau, Floradora and Cinnabar. Alas, there are no convenient markers to select rose dihaploids so just I treat my own few roxie progeny as being potential dihaploids.

Hi Don!

One time bloom is a useful dominant marker and is what has helped me identify some dihaploids in rose (Rise N Shine as a female in particular). It isn’t as early or convenient as being able to identify coloration or lack thereof in the seeds. In my work with potatoes I also used an inducer with a purple marker. The seeds of potato are somewhat translucent and we could look for purple hypocotyls in the embryos on a light board.

That sure is a reasonable suspicion in that some of these wide crosses that do not clearly have traits of the male may be from unfertilized eggs. There is evidence in the potato literature that many dihaploids have some DNA of the inducer. Fertilization may occur and there may be a bit of somatic recombination, but then the cell kicks out most but sometimes not all of the male DNA. There are the oat/maize addition lines that are great examples of that. Wide crosses with oats and maize pollen has led to haploids forming. Sometimes the oats do not kick out all the maize chromosomes and some come along. Eventually researchers obtained oats with most if not all of each of the 10 maize chromosomes separated in each line. So, oats plus chromosome 1 of maize, oats with chromosome 2, etc. This has led to a great tool to better understand the genes on each chromosome because of its effect on the oats.

It would be interesting to learn if Floradora and others have a little bit of R. roxb. DNA.

David, do you think it would be possible to use coloration of the freshly germinated hypocotyl as a marker in roses? I often notice distinctions in the anthocyanin expression in the developing embryos. Likewise, root branching is often distinctive.

I got a R.roxburghii (not f. normalis) this winter. Is R.roxburghii’s pollen easy to collect?

That’s a great question Don. Hopefully it could work if it is dominant and the roses we want dihaploids from don’t have that trait and the pollinator we use is homozygous dominant for the trait (or at least pretty close to it with multiple alleles if it is a polyploid).

Don (would be possible to use coloration of the freshly germinated hypocotyl as a marker in roses) I use this every year as a means of determining whether or not a cross has worked for me. Certain pollinators I use, produce seedlings with dark coloured hypocotyl, this ensures me that the part of pollinators genome has been transferred accross to the seedling. It is the first thing I want to see as it breaks through the soil.

Warren

Warren, which roses tend to produce a darker hypocotyl for you?

It looks like we need to start tracking hypocotyl coloration in order to establish carriers and their zygosity.

Don : Thank you for posting this video, it gave me a few ideas.

David Z: the cultivars which display this are two of mine, Arjuna and Kabookie.

Induced haploids are not always “pure”. This can be a problem in some cases, where certain clones of the “inducer” pollen parent leave a “genetic footprint” in the haploids.

Straadt and Rasmussen (2003)" “Introgression of DNA in the S. tuberosum cv. Pentland Crown dihaploids, after pollination with S. phureja IVP48 and S. phureja EC90, was demonstrated by the detection of 14 of 68 IPV48-specific markers in the dihaploids. However, no DNA introgression was found in any of the 30 S. tuberosum dihaploids derived from S. phureja IVP101.”
http://onlinelibrary.wiley.com/doi/10.1046/j.1439-0523.2003.00878.x/abstract

Bingham (2009) reported on hybrids of Medicago sativa X M. arborea.
“Interspecific and intergeneric hybridization does not always result in balanced hybrids with the chromosomes of both parents. Often there are changes such as chromosome elimination, chromosome duplication, chromosome fragment loss or gain, gene inactivation, gene activation, and activation of transposable elements (see literature reviews in the following references). There seems to be precedence in the literature for every possible outcome. Herein, we cite examples of DNA introgression and trait transfer in wide crosses without balanced hybridization. This appears to be the case in the sexual hybrids of Medicago sativa X M. arborea produced in Wisconsin, and in Australia. We have known for five years that M. arborea traits have been transferred. More recently, it has been shown that hybrids contain M. arborea-specific DNA . No two progeny are alike in fertility or morphology, and most contain different M. arborea-specific DNA bands. Thus, individual hybrids have pieces of the M. arborea genome, and the complete genome could be spread over the ca 20 hybrids on hand. This appears to be an advantage in backcrossing desired traits, because linkage drag is minimized.”
http://www.medicago-reports.org/volumes09.html

And again, Faure, et al. (2001) wrote similarly:
“Hybridisation between the annual diploid sunflower (Helianthus annuus) and the perennial diploid species Helianthus mollis and Helianthus orgyalis was studied. Cultivated sunflower was either used as female or male parent. Progenies were obtained by means of a normal crossing or embryo rescue procedures, but hybridisation success was low. All plants examined cytologically appeared to be diploid. Plants showed a great morphological variation, but they resembled the female parent-type predominantly. In crosses with cultivated sunflower pollinated by one of the two Helianthus perennial species, 35% of the plants had a sunflower phenotype. We applied RAPD and RFLP markers to determine the genetic constitution of progenies. It appeared that the contribution of the wild male parent was reduced in comparison with the female parent: hybridisation occurred at different levels depending on the plants. On average, only 5% of the minimum number of expected RAPD and RFLP bands from the male parents were recovered in plants produced from mature seeds after pollination of sunflower by H. mollis.
http://www.agr.unipi.it/dbpa/sunbio/Html/abs4.htm#O041

This is apparently what happened with Tantau’s hybrids. According to Wulff (1954):
“Without going into details I may state here that the three roses ‘Floradora’, ‘Käthe Duvigneau’, and ‘Cinnabar’, as well as the hybrid 46534, did not show any traces of the male parent Rosa Roxburghii in their morphology. The first three roses are true hybrid polyanthas, the latter is a true hybrid tea, indicating thus that the genes which are responsible for the respective characters of growth habit and for many characters of shape and size of flowers, fruits, leaves and spines are dominant to the allelic genes of R. Roxburghii. Only anatomical studies revealed a certain similarity and relationship to the latter species.”
http://bulbnrose.x10.mx/Roses/breeding/Wulff/Wulff_roxburghii.html

Then there are the “impossible” intergeneric orchid hybrids called Phragmipaphium (Phragmipedium x Paphiopedilum) which appear to have chromosomes from only parent, but traits from both.
http://www.google.com/images?client=safari&rls=en&q=Phragmipaphium&oe=UTF-8&hl=en&sa=X&oi=image_result_group&ei=QjWoVMjBG4anyQTbmIKoAw&ved=0CBYQsAQ

It would have been nice of Wulff to give us more details about the anatomical studies.

Hurst (1928) made another interesting comment regarding our Roxburghii.
Platyrhodon microphylla, from China and Japan, the so-called Chestnut Rose with cup-shaped fruits covered with fleshy spines, seems more promising in its fertility since I have succeeded in raising the second generation of a cross with Rosa rugosa. The results, however, although extremely interesting from the scientific point of view, are not very promising horticulturally, since the grandparent R. rugosa has been reproduced in facsimile several times, while the others resemble the parent hybrid with strange mutational variations.”

The hybrid ‘Walter Butt’ does look like a “proper” or balanced hybrid, unlike Tantau’s seedlings. But Hurst’s observation suggests that much of Roxburghii got left behind in the hybrid he studied. Otherwise we should expect some “facsimiles” of Roxburghii to turn up in the F2.
http://bulbnrose.x10.mx/Roses/Rose_Pictures/W/walterbutt.html

…“indicating thus that the genes which are responsible for the respective characters of growth habit and for many characters of shape and size of flowers, fruits, leaves and spines are dominant to the allelic genes of R. Roxburghii. Only anatomical studies revealed a certain similarity and relationship to the latter species.”

He would have to be wrong about that, of course, and here’s further evidence of mixed dominance and/or possibly partial or incomplete dihaploidization: two seedlings from pollination of roxburghii normalis by one of the French hedge-clipper roses.


True! Wulff wrote long before anyone understood how snippets could move from one chromosome to another. It is pretty clear that roxburghii did not contribute a complete set of chromosomes to Tantau’s seedlings. So dominance would not be involved. In fact, it is doubtful that even a single complete roxburghii chromosome made it in. The remaining maternal chromosomes then (apparently) doubled up to produce the tetraploid complement.

It would be interesting to backcross one of the seedlings to ‘Baby Chateau’ to see how (or whether) the anatomical differences were inherited.

I do wish I had had a chance to see ‘Baby Chateau’ to compare it with Tantau’s three seedlings (which I have seen).

Baby Chateau can be seen in person for the price of a ticket to Průhonice. I suggest that we lobby the management here to spring for the cost of sending me there to sniff it and take photos - or maybe someone has relations in Europe who could be our proxy in that regard?

[quote]Hurst (1928) made another interesting comment regarding our Roxburghii.
“Platyrhodon microphylla, from China and Japan, the so-called Chestnut Rose with cup-shaped fruits covered with fleshy spines, seems more promising in its fertility since I have succeeded in raising the second generation of a cross with Rosa rugosa. The results, however, although extremely interesting from the scientific point of view, are not very promising horticulturally, since the grandparent R. rugosa has been reproduced in facsimile several times, while the others resemble the parent hybrid with strange mutational variations.”

The hybrid ‘Walter Butt’ does look like a “proper” or balanced hybrid, unlike Tantau’s seedlings. But Hurst’s observation suggests that much of Roxburghii got left behind in the hybrid he studied. Otherwise we should expect some “facsimiles” of Roxburghii to turn up in the F2.
http://bulbnrose.x10.mx/Roses/Rose_Pict … rbutt.html
[/quote]

I played some along this path beginning more than ten years ago first growing Walter Butt: if very rugosa like and quite fertile it has fierce spines on fruits from rox amplified in some progenies I disregarded.
Then putting rox pollen (from an homogenous lot of seedlings grown from Quarryhill Botanical Garden chinese collection seeds) on other fertile rugosa derived plants. F1s are as tall erect divergent as is rox and fertility is very low. I got a few F2s using F1 pollen on other rugosa derived fertiles of which one has limited fertility and will be used further. F2 plants are also very very rox looking: flower, foliage, stems, architecture.
At the moment my opinion is that Walter Butt may be is not a F1 but a reverting to rugosa back cross.

Burdick (1951) had some strange results raising seedlings from doubled haploid tomatoes. The original haploid had fruit with colorless skin, but some of the seedlings had yellow skin. “Other plant characters gave some indication of segregation also.”
http://tgc.ifas.ufl.edu/vol1/v1p4.html

The obvious (though not necessarily correct) explanation is that some of the segregating characters involved changes in methylation (gene silencing). Perhaps the “gene for colorless skin” is really a silenced version of the “gene for yellow skin”. The shock induced by the change from haploid to diploid might be enough to reduce methylation overall, and thus “liberate” some of the silenced genes.

Would this occur in roses?

One should not assume too much about marker traits. Halsted (1904) assumed that the pollen parent always controlled the color of seeds in maize. He crossed ‘Black Mexican’ and ‘Country Gentleman’ in both directions, but discarded black kernels of ‘Black Mexican’ and white kernels of ‘Country Gentleman’. The progeny of BM x CG (raised from white kernels only) were more variable than from the reciprocal cross (black kernels only).
http://bulbnrose.x10.mx/Heredity/BlackMexicanGentleman/BlackMexicanGentleman.html

The dark color of the ‘Black Mexican’ is due to the presence of a transposon that is normally silenced. Apparently this silencing extends to some other gene or regulator that controls when pigment will be produced. White corn generally has pink or light red glumes, anthers and silks. In BM, to the contrary, those structures are white or pale green – the pigment (chrysanthemin) is produced in quantity only in the kernels. The “shock” of crossing with a different strain releases the transposon and adjacent genes from silencing, in many cases, leaving the kernels white or white with dark dots.