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Mutations from ultraviolet light.

Posted by Walter_Pickett 5-6 KS (My Page) on
Mon, Jan 24, 05 at 21:34

Does anyone know how much ultraviolet light is needed to make mutations in pollen? I have read of it being used, but I read it years ago in school. 25 years, and it is a bit fuzzy in my mind.
The reason I want to use ultraviolet light is that it makes point mutations without messing up the chromosomes.
And pollen is the only reproductive tissue that the untraviolet light will reach.

My purpose is to get a lycopene pink arilbred iris. The lycopene pink is recessive in tall bearded iris (autotetraploids) and in standard dwarf bearded iris (allotetradloids). The arilbreds are a different allotetaploid from the standard dwarf bearded, and no lycopene pinks exist in them.
My plan is to use arilbred pollen from arilbreds that have the lycopene gene in them from the tall bearded side of their ancestry. I'll irradiate the pollen with the light, then use it on the standeard dwarf bearded with lycopene. The mutations in the lycopene gene should give hybrids that show the lycopene color.
The arilbred x standard dwarf bearded F1 iris are sometimes partially fertile. This should get the mutation into the arilbred iris.
Any comments on this are welcome. Especially on how much and the type of ultraviolet light to use.
Walter


Follow-Up Postings:

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RE: Mutations from ultraviolet light.

What a great idea Walter. I wish I knew how much. I think there is more information out there about standard radiation and pollen exposure. For instance in roses irradiated pollen was used to induce seed formation without fertilization for eggs to generate into haploids. Many seeds though were not haploid as well and were from normal fertilization. Their dose was in the range of 250-2000 Gy of gamma rays using a cobalt source. 500 Gy worked about the best. Perhaps if you know a doctor he/she may be able to provide a similar dose with X-ray for you.

I've tried to us UV light to induce mutation in chrysanthemum, but didn't get much. I had callus growing in the dark (it was white without anthocyanin or chlorophyll because those things can help protect the cells from UV light). I then exposed the cultures for a few days and then regenerated plantlets and looked for mutants. I only found one red mutant from a purple clone out of over 1,000 regenerants. That UV source was a lamp used for looking at DNA in agarose stained with ethrydium bromide and the cultures were ~10 inches below it. Gamma radiation seems to hold more promise. I would like to do a similar thing as well to draw out recessives with other plants.

Good Luck,
Sincerely,
David


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RE: Mutations from ultraviolet light.

Thanks for the information.
I can get things irradiated at Kansas State University. It is generally expensive, but doable.
I chose to use UV because it generally causes point mutations, and not chromosome damage.
The F1 between an SDB and an AB has low enough fertility already. I don't need chromosome damage for this experiment.
And using the pollen has several advantages. One is that after this year, I can use stored pollen. Iris pollen can be dried and stored frozen for years.
But this is the first time I'm trying this, and I have no pollen stored. So I need something that will work overnight, as I need it. I can't be looking for someone to irradiate pollen for me on a moments notice.
If I have enough pollen to save for next year, I can ask the folks at KSU to stick the pollen in when they are doing some gama irradiation anyway, at their convience.


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RE: Mutations from ultraviolet light.

Walter

I dont know exact wavelengths you are looking for but I have an idea you might try if you are willing to experiment with time exposures.

Find someone with a UV sterilizer for ponds or water. Im sure one could be adapted.

I had an old UV hood I actually got from KSU's trash but threw it out when I moved to go to school at NCSU (I worked for Dr Clayberg too).

Sounds interesting what you are trying.

Good Luck
Keith


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RE: Mutations from ultraviolet light.

I have now found information about UV light and mutations.
UV C, from germicidal lamps, ionizes thymine. That's the T in the T, A, G, C, genetic code. While the thymine is ionized, reactions can take place that nutralize the gene.
So an exposure that is sub-lethal can make lots of mutations. Most will be recessive, which is just as well for my present problem.
Walter


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RE: Mutations from ultraviolet light.

You will have to screen many, many, many offspring. Theis is a method used, although I have never personally done it. Point mutations do "mess up" the chromosomes. I assume you mean point mutations that do not impact viability. You will get mostly normal offspring (no mutation or repaired damage or recessive)- some will never germinate because too damaged (point mutation in essential gene), and some will grow with point mutations - but mutations will be distributed over whole genome - every gene of thousands of genes - with only two or a few more genes associated with flower color. Dosage is an issue, as is wavelength. I think there are chemical mutagens that have been used for this purpose, so you might want to research it a bit on internet.


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RE: Mutations from ultraviolet light.

Brenda
Thank you for your comments.
I assure you I have researched this. As a former professional plant breeder, I am used to screening thousands of plants from a cross. I have not used mutagens before. But the screening shouldn't be harder than growing out a humongous F2
UV does mess up chromosomes some. But compared with harder radiation, the chromosome damage to mutations ratio is low.
I looked at chemicals. I ruled them out because the pollen dies if wet. Amonia fumes have worked on pollen, and a few other chemical fumes. But controling the concentration of amonia in air, to control dosage, looks more difficult that UV.
And the given gene I want to mutate will already be in the recessive state in 3 of the 4 genomes. In other words, it will be Aaaa. I want to knock out the A in the cross aaaa x AAaa. The pollen I will be irradiating will be all Aa.
Both these parents are amphiploids, and the amphiploids have one genome in common. The cross of these two amphiploids gives F1 plants that are somewhat fertile. Some of the F1s are somewhat fertile.
So when the F1 plants bloom, the aaaa plants, if any, should stick out like a sore thumb.
Thank you for the critique of my idea. It is though the comments of people like you that I am fine-tuning my experiment.
Walter


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RE: Mutations from ultraviolet light.

So these are tetraploids with disomic inheritance? (Otherwise all this mutagenesis stuff would seem kind of silly...)

Dose rates on these sort of things tend to be rather species and genotype specific anyway, so it would seem the best bet might be to try differing doses on different batches of pollen. This may not be a workable solution if you're limited in the amount of pollen or flowers at your disposal, I guess.

I dug through the papers I had on hand on mutagenesis, but did find much on UV...it doesn't appear to be a particularly favored method, for whatever reason (it's somewhat less penetrative, for one thing). All I found as far as a optimum dosage was 125 J/m2 for apple and pear leaf explants in tissue culture. I don't know that that has much bearing for iris pollen, but it'd at least be a starting point.


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RE: Mutations from ultraviolet light.

Thank you. That is a start.
In general dry tissue needs higher doses. I think pollen would count as dry, though it is ready to grow, it isn't dormant.
Yes, unless I can find a paper on irradiating pollen, I will have to try a wide range of doses.
In a week or tow, I'll be taking a day off to go to the KSU library to spend a whole day just looking through old journals looking for info on UV light and mutatins.
It seems that using UV on pollen had a short popularity. In most contexts, using anything on pollen meant looking in the second generation from the treatment, and growing several plants from each seedling from treated pollen. I can see why it isn't popular.
Walter


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this was rejected first time.

"So these are tetraploids with disomic inheritance?"

Actually, I am going to try both pink autotetraploids and the pink allotetraploids, both pollinated with pollen from the arilbbred amphiploids. But in both cases, the seed parent is homozygous pink. In both cses, the F1 has very low fertility, but does sometimes set seeds or give fertile pollen. So both cases are the same from a breeding point of veiw. In fact, in the progeny of the autotetraploid tall bearded iris x ampliploid arilbred and the standard dwarf bearded amphiploid x arilbred amphiploid, the F2 progeny get complete genomes, so the backcross progeny will be the same regardless of pedigree.
I checked with iris breeders about which cross gives more seedlings and the percent fertility of the seedlings. None could give me numbers. So I decided to try both and keep count of the numbers for future reference.
One way the amphiploid vs. autoploid does make a difference is that the amphiploid is much earlier to bloom than the autoploid. This is because the speices used to get the amphiploid is extremely early.
Walter


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RE: Mutations from ultraviolet light.

Walter,

I don't know if this will be useful to you, but Dr. Peter Werckmeister wrote in Iris colors and pigments, Bull. Amer. Iris Soc. no. 158: 25-33 (1960):

"Now I have found that it is easy to extract the yellow pigment with alcohol, but lycopin is insoluble in alcohol and is not removed. If then orange-colored beards are immersed in alcohol the yellow color is removed and they turn pink if they contain lycopin; if no lycopin is present they become white. Also, plants heterozygous for the tangerine gene, which appear among F2 progenies are of T1t3 genetic constitution, can be detected by this test. Thus such tests are important for the breeder or geneticist because in some irises with bright orange beards the color is due entirely to orange colored carotenoids."

He also wrote Lycopin and pink-breeding in The Iris Yearbook, the British Iris Society, p. 155-159 (1958), but I don't have that article.

Karl


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RE: Mutations from ultraviolet light.

Thanks Karl.
I recieved similar informatin on one of the iris groups I'm on. But I appreciate you're help, too.
I will already know the hybids I'll be working with will be T1t3 because of the cross.
But I'll loose that when I backcross to the arilbred amphploid. That is, unless I can identify a mutation which would then be t4. And that should stand out.
Now that I think about it, I could use Werckmeister's method of identifying T1t3 amphiploids in arilbred x arilbred amphiploids, using irradiated pollen. And I could cut out the low fertility SDB x arilbred F1. That would be a help.
Not all F1s from SDB x arilbred have any fertility, and I know that making the cross, and getting the mutation I want doesn't guarentee that I'll be able to go to the next generation with that plant.
You've given me a new angle to think about.
Walter


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RE: Mutations from ultraviolet light.

Walter,

I just think it's useful to remind the beginners who read these posts that "recessive" doesn't necessarily mean that a trait is not expressed at all. In many cases the recessive trait is present but masked. Tricks like using alcohol to remove alpha-carotene so the lycopene can be seen are very useful.

One time I crossed a purple rose (cyanin+co-pigment) with an orange (pelargonin). Pelargonin was present but masked because there was so much less of it and because the cyanin complex is so much darker in color.

There are other such tests, such as placing a flower over an ammonia solution. This is from W. J. C. Lawrence, The Genetics and Cytology of Dahlia Species. J Genetics, 1921:

(c) Reactions of anthocyanins and flavones with ammonia and SO2

As indicated on p. 128 the change of colour occurring when white, ivory or yellow petals are fumed with ammonia is as follows:

Petal colour---Changes to
White---No change
Ivory---Lemon yellow
Yellow--- Intense orange

The ammonia test is a well-known method used for detecting the presence of anthocyanin in plant tissues. If anthocyanin is present a green or bluish colour usually develops upon fuming, though possibly other substances in the cell sap may modify this green or bluish appearance. In addition to the many fumings made with ammonia in the course of these experiments, petals have been bleached with SO2, and the reactions of different flower colours with these two reagents noted.
The observations may be briefly summarised as follows:
SO2. SO2 does not affect the flavones. It half bleaches the deeper coloured petals and almost entirely bleaches tinged varieties. Penetration is increased if the petal is first fumed in ammonia and then bleachedcomplete removal of the anthocyanin pigments resulting, thus revealing the flavone ground.
Ammonia (a) Magenta and purple flowers give green and bluish-green reactions. (b) Orange and scarlet give an intense reddish-brown coloration. (c) Intermediate forms give intermediate reactions.
The intense reddish-brown which develops when orange or scarlet petals are fumed is of some interest, since this is not typical reaction of anthocyanin with ammonia.
All the other species I have grown conform, after their kind, to the above results.
=======

Pale ivory can look white, but the ammonia test reveals the difference. This, too, is an important consideration. "Modifiers" can influence the expressions of traits, even to the extent that "recessive" traits become dominant -- or vice versa.

I suppose you are aware of the chilling requirement of arilbred embryos. TB embryos lack this requirement. I don't know about DBs.

It is also interesting to note that some of the DB species have deep yellow flowers but lack carotenes. They are pigmented by chalcones. (I wish we had yellow chalcones in roses. Then we could have bright, non-fading yellow AND rich fragrance.) This raises the possibility of hybrids with both lycopene and chalcone -- maybe a bicolor with chalcone yellow standards and lycopene pink falls.

Even without mutations, genes can be swapped between genomes occasionally. That is, a Pumila gene may be transferred to a TB chromosome once in several hundred seedlings of tetraploid IBs. The same should happen, probably as rarely, in Arilbred varieties.

Karl

Here is a link that might be useful: Dominance Modification


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RE: Mutations from ultraviolet light.

Karl wrote "Even without mutations, genes can be swapped between genomes occasionally. That is, a Pumila gene may be transferred to a TB chromosome once in several hundred seedlings of tetraploid IBs. The same should happen, probably as rarely, in Arilbred varieties. "

Much more rarely.
The I. pumila and the tall bearded iris are both in the eupogon sectiion.
The arils are in a seperate section. Or subsection, I forget. But they are farther from the tall bearded than the miniature dwarfs are.
Even so, yes, I think that rarely gene exchange would take place.
I don't need an exchange though. Either a deletion or an exchange would be fine with me, if it gives the result. And I believe either would.
Generally, an exchange where pairing is very low is induced by hard radiation. That breaks up the chromosomes and pieces can end up in the wrong chromosome. And pairing is generally reduced.
A deletion, or more accurately perhaps, an inactivation, by changing a base pair, is more likely facilitated by UV or chemically induced mutation. And fertility is generally reduced less than with hard radiation.
The more I study it, the more I like UV on pollen. Relatively safe to handle. No fumes, it doesn't go through shielding. Turn it off and it is off. I'm expecting point mutations, with minimum fertility problems.
I found a germicidal UV light for $180. And maybe one for $50. Both would require modification, but they are possible.
The biggest problem I now see is that the treated pollen will give lots of other mutations, including recessive lethals. I'll be backcrossing to non-treated aribreds to keep or regain vigor.
Walter


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RE: Mutations from ultraviolet light.

Walter,

What I had in mind were translocations rather than exchanges or crossovers between homeologous chromosomes. This, apparently, is what happens when Pumila genes (or chromosome segments carrying the genes) are translocated to TB chromosomes.

The crossovers resulting in translocations are more likely to occur within heterochromatic regions, so the homology of chromosomes is not a factor. All heterochromatin is effectively "homologous", according to Prokofyeva-Belgovskaya, who observed "Mechanical weakness of their chromonemata, increasing the frequency of chromosomal rearrangement and crossing-over in them".

Certainly, hard radiation could fragment chromosomes resulting in rearrangements. However, what I described has been reported as occurring among seedlings untreated tetraploid IBs. I'm not discussing this as an alternative method, but as an alternative explanation.

Once you get the result you're after (I have my fingers crossed for you because I'd love to see a flamingo pink Arilbred) it would be interesting to observe the linkages, if any, to see whether you got a point mutation or a translocation.

Karl

Here is a link that might be useful: Prokofyeva-Belgovskaya


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RE: Mutations from ultraviolet light.

Karl
I think I understand now what you were saying and why you were saying it.
I guess the reason I didn't catch it the first time is that, while it might be useful in the long run to mix the TB and aril DNA more, giving more disease resistance and other garden tratis from the TBs. But right now I am trying to get a recessive gene, or almost recessive gene, into the aril chromosome. Getting another copy of the recessive gene into the aril chromosomes, without removing the dominant gene, I think, will be a waste of time. I need to get rid of the dominant.
That is my opiniion. Noted arilbred breeders, with good histories of success breeding arilbreds, and with gene and pigment studies, disagree. Some don't even agree that the evidence for a single recessive inheritance is convincing. I respect the opinion of those breeders. They have been successful in improving the arilbred iris, and I haven't. They have experience in trying for a lycopene pink arilbred, and I don't.
But 50 years of trying for lycopene pink arilbreds hasn't been sucessful. So I am on a diferent track, trying to knock out the dominant non-pink gene.
Should anyone want to read the discussion about this, it is on the aril Yahoo group. The name is aril robin or some such. There can't be that many aril Yahoo groups.
But I am getting different feedback here, and I apreciate it.
The reason I said lycopene pink above is that there are some nice anthocyanin lavender-pink arilbred iris. They are nothing to sneer at. But lycopene would give another color to the breeder's pallet.
And the anthocyanin and the lycopene would not be influenced by the same genes that determine the pattern of the color on the flower.
Having both would hugely increase the types of patterns available.
And in adition to the Lavender-pinks, there are aril and arilbred iris with ruby-red signal spots at the base of the beards already. This without lycopene.
Over 50 years since C. G. White made the arilbred iris a fertile, growible garden plant, and the variation is enormous. But the potential is hardly touched.


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RE: Mutations from ultraviolet light.

Walter,

Of course, a translocation of the "gene for yellow" (and linked genes) from the Aril chromosome to a TB chromosome would amount to a deletion since the extra "gene for yellow" would segregate out in some back crosses to a flamingo pink TB.

You startled me a bit when you mentioned that lycopene had been available in irises for 50 years. It can't be that long because I remember the early ones and ... Oh, right! I have been around that long.

My first flamingo pink was 'Happy Birthday', which was given to me by an iris breeder when I was about 4 years old. I still love the color.

Karl


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RE: Mutations from ultraviolet light.

Walter,
I just found this site looking for info on irradiation of pollen. I want to work with daylilies. I have a copy of a publication that gives some info.
POLLEN RADIOBOTANY
Radiation Botany 1962
Vol.1, pp101 to 154

It states good dosages are 5x10/5th,8x10/5th and 1x10/6th ergs/ cm2.

I have a Ray-tech versalume with an output of 4w but the manufactures can't tell me how far to place the pollen in order to get the dosage needed. I can't afford a radiometer.

This info may be too late but thought I would post.

Ira


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RE: Mutations from ultraviolet light.

This article (pdf) in a Swedish gardening magazine might be of some interest, all the plants pictured were created by a hobby-gardener using UV-radiation of seeds, seedlings and pollen.


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RE: Mutations from ultraviolet light.

Sure makes me wish I could read Swedish.

Could someone translate the method used by that hobby-gardener to get those cool plants??


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