Wet pollination and mutagens

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jbergeson
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Wet pollination and mutagens

Post: # 66946Post jbergeson
Fri Feb 02, 2018 2:28 pm

I have a friend who studies maize genetics and as part of his studies he treats corn pollen with ethyl methanesulfonate (EMS), a mutagen, in a mineral oil base. The pollen is then applied in the slurry of mineral oil.

Could this technique be applied to roses? Maybe some of the research people on here might have insight or be able to find relevant studies.

If the EMS is applied to the pollen, what base should be used? It seems that EMS is soluble in water but perhaps more soluble in oil. Some studies mentioned paraffin oil, while others used water. For some reason I am skeptical that an oil slurry of pollen would work well with roses. It seems like a wet pollination with water might work.

That raises the questions: has anyone used a wet pollination method for roses? How long will rose pollen survive in an aqueous solution?

Another option is simply soaking the seeds in a solution of the mutagen. It seems like it would be difficult to figure out how long to soak so the EMS passes through the achene to the embryo but doesn''t kill the embryos.

What genetic differences would there be between mutations that originated with treatment of pollen versus treatment of the embryos themselves?

It would be helpful if a researcher first established the viability of pollen that has been immersed in water or oil for various periods. Based on those results, they could then test various concentrations of EMS and soak times to find a treatment that kills about half of the pollen that would have survived a similar soaking in pure water or oil.

Another helpful preliminary test would be to compare hip set and seed quantity from pollen that is applied dry or in a various wet solutions (water, oils, maybe polyethylene glycol). This test could be done by any of us who have a lot of roses and the time to mess around.

It should be noted that EMS is a potent mutagen and carcinogen and any experiments with it should only be undertaken with strong isolation and protection protocols.

david mears
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Re: Wet pollination and mutagens

Post: # 66949Post david mears
Fri Feb 02, 2018 2:32 pm

Great question/s Joe. I guess maize pollen is larger, so the time frame for rose pollen would be shorter.
[color=#FFFF00]david mears
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Don
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Re: Wet pollination and mutagens

Post: # 67002Post Don
Tue Feb 06, 2018 12:43 am

I'd be interested to know from your friend just how effective mutagenesis is, overall, in his breeding program with maize. Does it give usable results that can be, have been, incorporated into commercial maize?
What doesn't kill them makes them stronger.

jbergeson
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Re: Wet pollination and mutagens

Post: # 67004Post jbergeson
Tue Feb 06, 2018 7:54 am

Hi Don! My friend is not a corn breeder, he is just studying genetics. So, no, I don't think he's accomplished any practical improvements with the method.

Don
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Re: Wet pollination and mutagens

Post: # 67007Post Don
Tue Feb 06, 2018 12:13 pm

Any breeders of commercial plants out there with experience with mutagenesis?

Most mutations are deleterious so generating a useful mutation with coarse mutagens is a numbers game. Site directed mutagens are a different story and the paper Henry posted recently is a road map for their use but Amazon isn't yet offering discounts on CRISPR.

Joe, didn't you put some effort into chromosome doubling with trifluralin or cholchicine? Anyone?

BTW it's the time to purchase crocus bulbs if you want to do cholchicine doubling, they tend to disappear from the stores off season.
What doesn't kill them makes them stronger.

jbergeson
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Re: Wet pollination and mutagens

Post: # 67009Post jbergeson
Tue Feb 06, 2018 2:14 pm

Don, I put quite a bit of effort into it for one or two years, and I still have a few plants that appear to be doubled seedlings of OP Rugosas, but they are disease-ridden and not terribly hardy so I've ignored them. And I've not pursued that strategy for many years.

But thanks for the reminder. The two areas in which it seems like it would be beneficial to double chromosomes are with rugosas and polyanthas. It seems like it would be valuable to have a tetraploid rugosa for integrating hardiness into modern roses without losing rebloom.

I would also love to have a doubled Darlow's Enigma, just for the fun of it. For some reason I can't get Darlow's Enigma to germinate in any quantity, so triflurilan doubling doesn't seem to be an option there for me.

Gotta match ambitions with time and energy levels. It seems unlikely for me to pursue mutagenesis at this point when it might be even more of a numbers game than chromosome doubling. But it is fun to dream.

david zlesak
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Re: Wet pollination and mutagens

Post: # 67010Post david zlesak
Tue Feb 06, 2018 4:44 pm

I'm not sure how long pollen needs to be in contact with EMS. Maybe painting EMS on stigmas (maybe putting it in a little bit of soft agar or gelatin to keep it moist) and then putting dry pollen on top of this would work. There still may be enough of the stigmatic exudate to allow the pollen to germinate (pollen germinates in a little bit of sugar water though too and a bit of sugar can be added to the mix) and then the pollen can germinate and then the tube itself grow through the mutagen. It may allow for greater contact of the nuclei with the EMS than exposure with the dry complete grains before they germinate and covered with the protein coat. Treating pollen grains will hopefully mutate the DNA in the nuclei before fertilization and then the embryo would have every cell with the mutation. If we wait until the seed germinates we may get a range of cells mutated and difficulty stabilizing it as the growing point eventually gets dominated by one cell type.

jbergeson
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Re: Wet pollination and mutagens

Post: # 67018Post jbergeson
Wed Feb 07, 2018 12:03 am

It's always great to get the input of a genuine practicing scientist, David! Thanks!!!

Do you think it would be more practical to attempt this with diploids rather than tetraploids? It seems like the mutated gene would have a greater chance of being expressed. Do you have any input on whether a mutation is more likely to be recessive and therefore might not show up until the next generation, making it especially important to work with diploids?

henry kuska
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Re: Wet pollination and mutagens

Post: # 67019Post henry kuska
Wed Feb 07, 2018 1:15 am

A number of years ago I used pollen dissolved (or slurried) in organic solvents to try to increase the efficiency of pollination. I wrote about those experiments in the old rose hybridizers forum. I did not think of adding a mutagen to the solvent.

This was the link:
http://www.rosehybridizers.org/forum/me ... =1757#1777
I do not know if someone can link to this message.
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jbergeson
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Re: Wet pollination and mutagens

Post: # 67052Post jbergeson
Sat Feb 10, 2018 3:13 pm

I thought of maybe a practical application for the use of a mutagen on pollen.

Treated pollen of R. nitida applied to a fertile reblooming rugosa such as Frau Dagmar Hartop. The specific mutation one would be looking for is a deactivation of the once-blooming gene of R. nitida. It should be easy to spot successful crosses, with R. nitida's shiny foliage, and if one of them ended up reblooming you'd have the desired result. That cross should produce a ton of seeds, which you'd need since this would be very much a long-shot numbers game.

I suppose the same could be done with R. nitida pollen on any reblooming rose.

Don
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Re: Wet pollination and mutagens

Post: # 67078Post Don
Tue Feb 13, 2018 1:46 am

I have some rugosa's here as part of the TAMU Rose Rosette Disease project - Frau Dagmar, Purple Pavement and Theresa Bugnet - and Magseed which I got from Paul Barden a decade ago as his recommended alternative to Linda Campbell. Based on my experience, admittedly as a novitiate, if you are going to embark on a mutagenesis program using any of them then get started asap so you live long enough to see results.

I have a question about nitida - why are the leaves shiny?
What doesn't kill them makes them stronger.

henry kuska
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Re: Wet pollination and mutagens

Post: # 67082Post henry kuska
Tue Feb 13, 2018 11:04 am

I had a gamma radiation cell in my office, and I also used trifluralin. Most of my mutated plants were monsters similar to what was found around Chernobyl, Russia. I did produce some tetraploids. One tetraploid rugosa was extensively distributed but seems to have dropped out of sight. A tetraplod acicularis (produced from the diploid form) gave an interesting number of seedlings when crossed with other roses but they all sooner of later died.

http://rosebreeders.org/forum/search.ph ... acicularis
http://rosebreeders.org/forum/viewtopic ... alin#p3049
http://rosebreeders.org/forum/viewtopic ... +61#p59393

jbergeson
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Re: Wet pollination and mutagens

Post: # 67083Post jbergeson
Tue Feb 13, 2018 11:49 am

I certainly don't have the energy to even think about embarking on a mutagenesis program this morning. I'm typing here as part of a procrastination strategy to avoid working on my spring catalog.

To answer Don's question about R. nitida, one spring morning, many centuries ago, Nitidus, one of the minor Gods of Wind and Light, woke up in the mood to travel. He sprang away from his palace on the island now known as Iceland and used his divine intuition (a primitive precursor to Google Maps) to guide him across the ocean to the sunny shores of Newfoundland. When grazing the local flora he came across a wild rose with a fragrance that was...divine. "The fragrance of this rose," he thought, "is simply divine. It should be named after a god like me."

"It's too bad the foliage is so lackluster in it's coloration," he mused. (Note: the prevailing view among modern theobotanistorians is that the rose Nitidus sniffed was R. blanda, named for its bland, unexciting foliage.) He inhaled a great breath and with an explosive sneeze pollinated many hectares of wild roses with divine pollen. The descendants of those roses now have shiny foliage and a compact habit. They form a low, suckering, deciduous shrub, growing up to a metre in height, although often less. Their stems are thin and covered in fine bristles. Their pinnate leaves have 7 to 9 shining leaflets which turn bright red, yellow and purple in the fall.

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