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Hybridizers: Chromosome Doubling

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Michael_Ronayne
Nutley, NJ
(Zone 6b)

November 1, 2009
12:06 AM

Post #7228416

OK, you have your F1 hybrid and it is everything you were hoping for but there is one small problem, the plant is sterile. Not a problem you say, you will just order some Colchicine and double the chromosome count:
http://wardsci.com/product.asp_Q_pn_E_IG0015182

But wait, unless you are a licensed research laboratory you canít order colchicine. So it is time to start thinking about Plan B. Before there was colchicine, there was Nicotine Sulfate which was used as an insecticide until it was discovered that it would double chromosomes quite by accident.

Test Tube Magic Creates Amazing New Flowers by Edwin Teale
Popular Science May 1940
http://books.google.com/books?id=iicDAAAAMBAJ&pg=PA57

Even today commonly and legally available agricultural chemicals such as oryzaline and trifluraline are being tested as colchicine alternatives, for their ability to double chromosomes.

Interspecific Hybridisation in Woody Ornamentals
https://biblio.ugent.be/input?func=downloadFile&fileOId=491800

Note: The herbicides oryzaline and trifluraline did not work for Hibiscus so donít waist your weekend reading the 190 pages of the above excellent PhD dissertation if you are interested in breeding Hibiscus.

Depending on the plant species, there are alternatives to colchicine. Can anyone report success with chromosome doubling? What is the current status of Nicotine Sulfate? Are there other alternatives to colchicine?

Mike

Zen_Man
Ottawa, KS
(Zone 5b)

November 3, 2009
7:25 PM

Post #7237792

Mike,

Many years ago I purchased a kit that included colchicine for doubling the chromosomes of plants as a home science experiment. I did succeed in converting a few young zinnia seedlings to tetraploids (or possibly even polyploids) by treating the growing tips of the seedlings. The kit had extensive precautions to take when handling colchicine and stressed its hazards. The kit itself contained a fairly dilute solution of colchicine. It was a long time ago, but I think the kit had you further dilute that stock solution for actual application to the plants. You treat the growing plants with about 0.1% to 0.2% solution, so a very small amount of the pure chemical would be sufficient for considerable experimentation.

I personally don't particularly like the results you get with tetraploid zinnias. They aren't resistant to mildew, they grow somewhat slower and have less branching of the plants. Their flowers do tend to be a bit larger, but not a whole lot. The flower form is affected, with spoon-like petals, not to my liking.

If I wanted to renew my experiments with tetraploid zinnias, or allotetraploid zinnias, I would pursue the acquisition of colchicine. The Internet has a lot of online stores, and some of them may not enforce the restriction to educational or research organizations. My zinnia breeding hobby is actually educational for me, and a form of research. You might join a hobby group like the Tissue Culture group, and try to obtain it through them. Or join an educational or research organization.

Colchicine is also a prescription drug for the treatment of gout-like symptoms, although it is probably more expensive in that form. I don't know for sure, but it might be that someone on the Internet would sell you small amounts of colchicine without any special affiliation. Here is a link to explore, and also see what you can find on eBay.

http://www.artchemicals.com/ProductDetails.asp?ProductCode=CO115-250MG&click=2

Colchicine is hazardous and is regulated by the FDA here in the United States. I was very careful when I was handling the stuff.

ZM

Michael_Ronayne
Nutley, NJ
(Zone 6b)

November 7, 2009
10:29 PM

Post #7251249

ZM,

Thank you for the feedback. It looks like Colchicine is very difficult to obtain in the USA. Nicotine Sulfate has also been band as an insecticide but you can still easily make the stuff for now; but, after looking at the properties of the chemical I donít know if I would want to work with it even though I have my advanced degrees in chemistry. I also suspect that as an unintended consequence of the war on tobacco, Nicotine Sulfate will soon be declared a Schedule One Controlled Substance. I have access to old research papers and will continue to look for legal and effective alternatives to Colchicine. Another possibility is making contacts at a local college which is doing research in this area but must instructions are not interested in amateurs.

There are several North American Hibiscus crosses for which the F1 hybrids are know to be sterile, on which I very much like to try chromosome doubling. If you come across a source of Colchicine keep me in mind as I would be interested in a coop deal. Is there any particular Tissue Culture Group that you would recommend?

Mike
Zen_Man
Ottawa, KS
(Zone 5b)

November 8, 2009
5:44 PM

Post #7253240

Mike,

"Is there any particular Tissue Culture Group that you would recommend?"

I'm a newcomer to this Tissue Culture thing myself. I chose the Home Tissue Culture Group because it is for amateurs.

http://www.hometissueculture.org/

"There are several North American Hibiscus crosses for which the F1 hybrids are know to be sterile, on which I very much like to try chromosome doubling."

If Hibiscuses grew faster, I would be breeding them myself. The Hibiscus gallery at Hidden Valley Hibiscus is fantastic.

http://www.hiddenvalleynaturearts.com/catalog.htm

But zinnias satisfy my desire for quick results. My fourth generation of zinnias this year are coming into bloom now, so I may have the opportunity to start a fifth generation this year. You can do a lot of breeding with zinnias in a relatively short time.

I asked Dr. Carol Stiff about the availability of Colchicine and she said they don't plan to offer it in the near future. But she did forward my query to the Home Tissue Culture Group and I did get a response from "Frankco" that said:

"I purchased Surflan from eBay. It's packaged in a smaller size, 8oz but it's quite enough to last a very long time. The cost is $18 plus shipping. The seller was very prompt with delivery.
http://tinyurl.com/yfk94y2

Surflan is 40.4% Oryzalin. Oryzalin is, in many cases, more effective than Colchicine, its safer to use and it's not as hard on the plants as Colchicine. You still should wear protect clothing such as goggles, and gloves when handling. Just follow the safety sheet."


I don't need to do any chromosome doubling yet, but I do plan to further research my options there. I haven't given up on the idea of purchasing Colchicine. I've never used Oryzalin, but it might do the trick. And there may be other alternatives to Colchicine as well.

ZM





Michael_Ronayne
Nutley, NJ
(Zone 6b)

November 9, 2009
1:48 AM

Post #7254592

ZM,

In the reference to the above PhD desertion both oryzalin and trifluralin failed to have any effect on Hibiscus although they were effective on Hydrangea and Buddleja (Buddleia). A search in Google Scholar indicates that Oryzalin is well regarded but direct research of the effects of Oryzalin on Zinnia was illusive:
http://scholar.google.com/scholar?hl=en&q=zinnia+polyploid+oryzalin+OR+trifluralin

There is quite a bit on zinnia polyploids:
http://scholar.google.com/scholar?hl=en&q=zinnia+polyploid

And colchicine has been the chemical of choice when working with Zinnia:
http://scholar.google.com/scholar?hl=en&q=zinnia+polyploid+colchicine
One of the problems with research papers is that many times they donít report failures. But unlike Hibiscus I did not find any report that oryzalin would not work for Zinnia.

Given that Oryzalin is legal (today), available and not that expensive, it is definitely worth a shot with your Zinnia. If I ever get the Hibiscus hybrids I am after and they are sterile I am going to have to do some serious searching.

I will follow up on the tissue culture groups. Thank you for your guidance.

Mike
starlight1153
Seale, AL
(Zone 8b)

December 30, 2009
1:22 PM

Post #7409223

Mike.. Thos e of us that hybridize Daylilies are constantly looking and working for ways to convert the dips to tets. Soem of the successful conversion are introduced.

Back in 2006 I wa s asking some of my friends if they had tried to work with oryzalin as this is a link I ha d found and ha d shared with them.

http://members.tripod.com/~h_syriacus/tetraploidy.htm

One hybridizer had used Oryzalin on abotu thousand Hemerocallis seeds and came up with 12 conversions. Another hybdridiers has soem succes s with it converting seeds to, but the conversion count again was very low, but to my way of thinking even if you get only a coupel of converted plants, and they are not worthy of anintroduction, they would at least be worth the hassle just to have the future genes.

At the time I was askign abotu the above link, I had also aske d this question because while the germinated seeds converted failrly easily, most died shortly afterwards in growth. Also when Oryzalin was use d in mist form on foliage it caused death of the seedlings.

"In herbicide clas s we had discussed Oryzalin and since it kills grass and small broad leaves, I wondered how could it convert Daylilie s which are like grass?

Also wondered how much difference is there between Surflan 4 AS , which has the Oryzalin, and Snapshot 2.5TG , which has Isoxaben 0.5% and Trifuralin 2.0%. Snapshot is supposed to be the first herbicide safe for around Daylilies and Hosta, but it still a herbicide and if the Oryzalin has capabilities for conversion, yet kills the seedlings, could Snapshot, which is over the top and first herbicide that safe to be used on wet foliage, do the same type of conversion on Daylily seeds, if treated with it maybe without killing the seeds? "

One of the responses I recieved was this. " My understanding is that the primary mode of action of both oryzalin and trifluralin as herbicides (particularly pre-emergent herbicides) is due to their effects on mitosis in the roots - they disrupt it. That is, also the reason why they can be used as chromosome doubling agents on plants. The primary difference in their use as herbicides versus chromosome doubling agents would be which tissues are being affected and the concentrations. Lower concentrations would be less likely to kill and not treating the roots would cause less death. When used for treating seeds that have sprouted most of the seeds will die because their roots have had their cell divisions blocked."

One of the things you have to take into with workign with conversions that was brought up to me was the fact that the converted plant may not be genetically identical to the parent plant other than doubled chromosomes. That some lose DNA, have smaller cells , and even though converte d may still switch off those all but one set of chromosomes.

I had so many experiments going at the time hat I never did get to try and work with the Snapshot2.5TG to see what if any effect it would have conversions.

You might try starting out using the Caffine conversion method first and see if that has any effect.

Here is some articles, copied from friend to me, as I always tryign to think outsid e the box and had been tryign to see if I couldn't try grating a dip and tet Daylily roots and crowns together after seeing how some trees when branches on the trees had merged as one when grown together for long period of time.

"Caffeine does not work the same way as colchicine in doubling chromosomes but it has been used successfully a couple of times.

This paper used a caffeine soak on wheat:

Genome Volume 40, Issue 4, 1997, Pages 552-558 "Chromosome doubling of haploids of common wheat with caffeine" Thomas, J., Chen, Q., Howes, N.

This one had caffeine in the tissue culture medium:

"Chromosome numbers of Saintpaulia ionantha plantlets regenerated from leaves cultured in vitro with caffeine and colchicine." F. J. Espino and A. M. Vazquez Euphytica 30 (1981) 847-853

This one injected caffeine into the flower buds to double the chromosomes in the gametes

Korean J. Genetics 27 (3) : 219-226 (September 2005) "Meiotic Polyploidization with Homoeologous Recombination Induced by Caffeine Treatment in Interspecific Lily Hybrids" Kim-Byung Lim , Rodrigo Barba-Gonzalez, Shujun Zhou , MS. Ramanna and Jaap M. van Tuyl

All three successfully doubled chromosomes with caffeine."



Now that I have throughly confused ya. LOL I ask this:

Mike... I not sure by doign a conversion that you would end up with some viable pollen. Having trouble wrapping still half asleep brain around it. Not sure if this will help you or not. We had a bit back discussion going over in the Brugsmania forum about sterile plants.
Think you might have better luck with mixing pollen. Not sure what other hybiscus cultivar you would use though, but you could experiment. Then check for pollen viablility before tryign to grow the pollen grains out.

Some long threads for ya to read through..

Start:

http://davesgarden.com/community/forums/t/920110/

page two:

http://davesgarden.com/community/forums/t/921479/

http://davesgarden.com/community/forums/t/924132/

http://davesgarden.com/community/forums/t/924059/

If you still want to get some Colchicine, and can't get any, pop over to the Daylily forum and make a post that you working on a project with a differnt type of plant and if anybody has a little bit they would be willing to share with you to dmail you. I know several of the hybridizers over on that forum and several have done conversions. Shoot folks have even been working at converting tets to hexaploids.













irisMA

irisMA
South Hamilton, MA

December 30, 2009
3:46 PM

Post #7409622

Since colchicine is such a dangerous product is it worth using? I did now that herbicide could be used in a successful doubling, never knew which one. Is it worth doubling the ch. I know since bearded iris are mainly tets we have a lot of colors etc not available in the diploids. Is that the way in daylilies? To the average gardeners eye are tet DLs worth it? I don't think we grow any, but have heard dispariging comments about them, especially about hardiness.
Zen_Man
Ottawa, KS
(Zone 5b)

December 30, 2009
9:13 PM

Post #7410675

Lucy,

"Since colchicine is such a dangerous product, is it worth using?"

Some people might not feel comfortable following the needed precautions. But if I want to double the chromosomes of some zinnias, then, yes, it is worth using to me. Actually, a few decades ago, when I was gardening in Fort Worth, I purchased a plant breeding kit that included colchicine. The restrictions were laxer then, but it did have explicit warnings and instructions for its safe usage. I did succeed in making tetraploids of several diploid zinnias, although the resulting tetraploids were disappointing. At that time I was going for really large zinnia blooms, and hoped that tetraploidy was a way to achieve that.

Colchicine needs to be handled with care, but several herbicides are at least as dangerous. Colchicine is a natural substance in the Autumn Crocus. Anyone who has been around those Crocus plants has been around Colchicine. At one time Colchicine was prescribed as a treatment for Gout. So there have been people who were popping Colchicine pills at the recommendation of their doctor. When I feel a need to double some zinnia chromosomes, I will seek a supply of Colchicine. And I will be careful with it.

ZM

irisMA

irisMA
South Hamilton, MA

December 30, 2009
10:08 PM

Post #7410861

Dr. McEwen how produced the tet siberian irises used it. But then he had been around it using it in the treatment of arthritis. L
starlight1153
Seale, AL
(Zone 8b)

January 1, 2010
8:54 PM

Post #7417353

I knwo I went looking for some the oen day and poppe din to see a professor friend and thankfully she had given me all the warnings. How if you get it on you it can be changing your cells and genetic s around. I personally don't feel comfortable enough workign with it, but with Mike's history I sur e he use d to workign with all them chemicals. Usually the colchicine doesn't work unles s ya injecting it or treating the crown.

If I want somethign treate d with it, there several well known , respecte d in the field hybridizer s that for a fee will treat your plants. Fees aren't to ba d either, not if it somethgin ya want really bad.

Lucy... I grow and work with both tets and dips. I have no problem with either one, but the avaerage gardner is not gonan knwo the differenc e between them. Your avg gardener goign for pretty. I love the new in roads made with the tets more color and genes . A fe w hybridizer s I knwo are not goign back to work with the dips. There still unknown possibilities to be found in them.

ZM... I been thinking abotu ya all week, sowing away on Zinnia seeds again. Nothign yet clos e to rival yours. : )





waltseed

December 30, 2010
3:40 PM

Post #8283925

My grad work was with crosses of wheat, rye, and barley. Doubling the chromosomes of the hybrids was a step we did research on. We found that it was best to keep the chochicine off the roots. Rather, we would use a sticky solution of agar and colchicine and put it in a petri dish. We would stick the top of the seedling into the agar, and turn the dish up-side-down and let the seedlings hang there. The roots would be uncovered, but in a 100% humidity atmosphere. After a day, we'd take the seedlings out, wash them well in running water, and plant them. This had a much better survival rate than methods where the cholchicine got on the roots. And who cares if the roots are doubled? The top part of the plants give the seeds.
We didn't develope this method. It has been over 30 years, and I don't remember who to credit. But it worked and I think it would work better on other chemicals as well.
Michael_Ronayne
Nutley, NJ
(Zone 6b)

December 31, 2010
8:35 AM

Post #8284882

Walt,

Thank you for a really interesting idea. Your timing was unbelievable. As I canít buy Colchicine legally, and the one published paper on using Oryzalin on tropical Hibiscus reported that it didnít work, I decided to give the original chromosome doubling chemical, Nicotine Sulfate, a try with my hardy Hibiscus.

Nicotine Sulfate (AKA Black Leaf 40) is no longer available in the United States and tobacco products canít be sent through the mails but there is an interesting free-market response to Americaís newest war-on-tobacco. As of December 31, 2010, you can still legally (I donít know for how much longer) buy tobacco leaf at local stores which sell a micro cigarette factory, which is about the size of a toaster-oven, that you can use at home to make cigarettes. Including your favorite blend of tobacco, the cost of consumables and the micro factory, a pack of homemade cigarettes is half the cost of a pack in New Jersey and a third the cost of a pack in New York. One pound of cut Virginia leaf is $20. Being a non-smoker, a pound should last for years if I freeze it in a freezer safe bag. If my tests are not successful, I can always turn my Virginia leaf into an organic gardening approved environmentally friendly [sarcasm] insecticide. The extraction of Nicotine Sulfate is an easy process involving boiling water.

I am using the Demo Method to germinate Hibiscus seeds and was planning to expose the hardy Hibiscus seeds to varying concentrations of Nicotine Sulfate until the seedlings are transferred to soil. I am still going to do that but your suggestion provides me with another option for just treating the leaves. In the original 1930's research of Bulgarian scientist Dontcho Kostoff, Nicotine Sulfate was only applied to the leaves of the plants which were infected with insects.

Were there any papers published on this technique? All I need is the name of principle investigator and the approximate date.

Thanks for the tip, Mike


This message was edited Dec 31, 2010 11:44 AM
Michael_Ronayne
Nutley, NJ
(Zone 6b)

December 31, 2010
9:16 AM

Post #8284932

Walt,

You provided enough information to get me started. Here is a 1955 paper on the agar- cocchicine technique know as the ďsmear treatmentĒ in this paper:

http://www.springerlink.com/content/v196333v34157232/fulltext.pdf

By seed treatment a rather high percentage of the seedlings are killed because of inhibition of the root development. This trouble is overcome by the smear treatment of young seedlings. The number of tetraploids, in percentage of seedlings treated by the smear method, may be as high as the number of tetraploids in percentage of seeds treated by soaking. But compared on the basis of surviving plants, the seed treatment has given a higher percentage of tetraploids. Usually only the plants which look most affected are chosen for breeding, and the rest is discarded without any further testing.

Recently Springer Link removed the green-wall and access to content is free. I donít know if this is permanent change or if only selected journals are available but the change is welcome because I donít have to run to the local university library quite as often.

Mike
Zen_Man
Ottawa, KS
(Zone 5b)

December 31, 2010
12:23 PM

Post #8285206

Mike,

I'm not optimistic about your chances of success with nicotine sulfate. As a kid on a farm, we sprayed a lot of Black Leaf 40, as a contact insecticide, on a lot of different plants. We killed a lot of insect pests, but as far as I know we didn't create any tetraploids or polyploids.

Colchicine has its hazards, that require care in its use, as do many laboratory reagents. It has been used successfully with Hibiscus chromosome doubling, as was stated in the thesis that you linked to originally. There are restrictions on purchasing and shipping colchicine, as this link shows.

http://www.phytotechlab.com/Detail.aspx?ID=761

It sounds to me like you are planning to do near-research-grade experiments with Hibiscus and that you would be capable of observing the rules for handling colchicine. I am sure that if you were doing your Hibiscus research for a University, you would have no problem getting the colchicine. In fact, it may be that you could purchase colchicine now if you could just have the colchicine shipped to a "non-residential" address.

ZM


This message was edited Dec 31, 2010 2:48 PM
Dave67
New Port Richey, FL
(Zone 9b)

December 31, 2010
10:27 PM

Post #8286033

Hi Mike, I have an interest in this subject and was glad to see you started this conversation.
I grow a plant that also contains colchicine. It is called Gloriosa superba.
Here is an excerpt I found about this plants properties...

The medicinal importance of Gloriosa superba is due to the presence of alkaloids in all parts of the plant, mainly colchicine (superbine), an amino alkaloid derived from the amino acids phenylalanine and tyrosine. The presence of colchicine-type alkaloids with a tropolone ring is characteristic for most genera in Colchicaceae. The seeds are the best source of colchicine, as their content is 2Ė5 times higher than in the tubers. A report from Rwanda claims that the highest colchicine content is found in young leaves.
Several colchicine-related alkaloids have been isolated from tubers and seeds. They are mostly demethyl substitutes and include cornigerine, which is a potent antimitotic, and colchicoside used as a muscle relaxant. A plant can contain up to 0.9% colchicine and 0.8% colchicoside.
In medicine, colchicine is used in the treatment of gout. In spite of its serious side-effects it is still commonly used for acute gout. It reduces the inflammatory reaction to urate crystals deposited in the joints. Its efficacy might be due to decreased leukocyte mobility. The substance is not an analgesic, and has no effect on blood concentration, nor on renal excretion of uric acid. Diarrhoea, nausea, vomiting and abdominal pains are the first signs of poisoning and occur at doses equal to or lower than those needed to treat gout. The diarrhoea may become severe and haemorrhagic. A burning sensation in the throat, stomach and skin may also be an early sign of intoxication. Nibbling on the tubers causes numbness of the lips and tongue and loss of body hair. Severe reactions include extensive vascular damage and acute renal toxicity with oliguria and haematuria. The patients may develop convulsions, delirium, muscle weakness, neuropathy and ascending paralysis of the central nervous system. In patients who have taken an overdose of Gloriosa superba tubers, death occurs as a result of respiratory depression and cardiovascular collapse within a few days.
Colchicine is a powerful antimitotic agent that blocks or suppresses cell division by inhibiting mitosis, the division of a cellís nucleus. It is used in plant breeding to induce polyploidy, as it allows chromosome division but inhibits formation of a mitotic spindle figure, which guides the separation of the two sets of haploid chromosomes. As a result no sister cells are formed. Once the treatment has stopped, however, the normal spindle figure forms again. Colchicine also inhibits the division of animal cells, but it is too toxic to be used to arrest tumour growth. A biosynthetic precursor of colchicine, demecolcine, has a wider margin of safety and is used to treat myelogenic leukaemia and malignant lymphoma. Extracts of the shoots and of the tubers of Gloriosa superba show strong nematicidal activity, which can be largely attributed to colchicine. The chemical constituents of the tuber are known to be very poisonous to fish. Severe damage is done by colchicine-containing plants to livestock in different parts of Africa.
In vitro production of colchicine is feasible, although the levels are in general 10Ė25 times lower than those found in plants grown in vivo.
Adulterations and substitutes
The corms of Colchicum autumnale L. and Iphigenia spp. (also Colchicaceae) are traditional sources of colchicine. An increase in demand for colchicine stimulated the search for an alternative source, leading to Gloriosa superba. Chemical synthesis of colchicine is possible but complicated. Synthesis is expected to remain an important target of the chemical industry. In-vitro production of colchicine is also possible but gives low yields.

excerpt from here: http://database.prota.org/PROTAhtml/Gloriosa%20superba_En.htm



This message was edited Jan 9, 2011 6:52 PM
Michael_Ronayne
Nutley, NJ
(Zone 6b)

January 2, 2011
10:30 AM

Post #8288279

ZM,

As of September 2010, obtaining colchicine just became a bit more difficult in the land-of-the-free:

FDA orders halt to marketing of unapproved single-ingredient oral colchicine
September 30, 2010
http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm227796.htm

The U.S. Food and Drug Administration today took action against companies that manufacture, distribute, and/or market unapproved single-ingredient oral colchicine, a medication commonly used for the daily prevention of gout, to treat acute gout flare-ups, and for the treatment of Familial Mediterranean Fever (FMF).

The companies are expected to stop manufacturing single-ingredient oral colchicine within 45 days and must stop shipping this unapproved product in interstate commerce within 90 days. A small amount of unapproved colchicine is expected to be available after these dates until supplies are exhausted.


This is of course great news for Merck, the manufacture of Colcrys, as the FDA has just put all of their generic competitors out of business. This is now you extend quasi-patent protection to naturally occurring products which canít be patented, just as long as you are willing to play ball with Washington. But medical users of colchicine should not worry, now that the FDA is in charge, you can still obtain colchicine with a prescription and a $25 co-pay. How much would anyone like to bet that generic colchicine was a LOT less than $25?

I am not sure what the impact will be on PhytoTechnology Laboratories but the long-term indications are not good. If colchicine is still available as a reagent it will not be for much longer and one should act quickly. My problem is that I donít have access to a non-residential mailing address to which I would be willing to have colchicine shipped; where will be too many warning labels on the package. With all those extra ďsafetyĒ charges, 1g of colchicine will cost $126.25 plus standard shipping rates; the 0.5g quantity is no longer cost effective. The bottom line is that if you have any interest in colchicine you had best act quickly.

Your observations about the relative effectiveness colchicine and nicotine sulfate are quite correct but I would still like to see exactly what the differences are and why colchicine became the reagent of choice. Dontcho Kostoff, who did the original work with nicotine sulfate in 1931, was by the late 1930ís using colchicine, acenaphthene and elevated temperatures to induce polyploidy in plants. Here are some Google Scholar searches:

http://scholar.google.com/scholar?as_q=colchicine&num=50&btnG=Search+Scholar&as_sauthors=Kostoff&as_sdt=1.&as_sdtp=on&as_sdts=31
http://scholar.google.com/scholar?as_q=acenaphthene&num=50&btnG=Search+Scholar&as_sauthors=Kostoff&as_sdt=1.&as_sdtp=on&as_sdts=31

A number of the papers in the above searches have free access to PDF copies thanks to the Indian Academy of Sciences. Of particular interest was this paper where Dr. Dontcho Kostoff compared different polyploidy agents:

Irregular Meiosis and Abnormal Pollen-Tube Growth Induced by Acenaphthene.
By Dontoho Kostoff, 1938
Academy of Sciences of U.S.S.R., Institute of Genetics, Moscow U.S.S.R.
http://www.ias.ac.in/jarch/currsci/7/8.pdf

Chemical agents like chloral-hydrate, chloroform, ether, alcohol, nicotine sulfate, lactic acid, etc., have been used for inducing irregularities in the mitotic and meiotic processes which might lead to formation of heteroploid and polyploid cells. More effective agents for this purpose are colchicine and acenaphthene. These two chemical agents reduce or completely paralyze the activity of the factors thatē condition the arrangements of the chromosomes into a regular metaphase plate (equatorially) and the formation of a regular spindle. In fact, these two phenomena are causally linked. In the previous publications I recorded some data upon the irregularities of the mitosis induced by these two agents. In the present paper I am giving some new data upon the irregularities in the meiosis and abnormalities in the pollen-tube growth induced by acenaphthene.

Google Scholar has known deficiencies with non-English searches so be careful and ask the same question several different ways. Also see the following links, many of which are not in Google Scholar:
http://www.google.com/#q=dontcho+kostoff

Today Dr. Doncho Kostoff is honored in his homeland:
The Acad. Doncho Kostoff Institute of Genetics at the Bulgarian Academy of Sciences.
http://ig.bas.bg/eng/index.php?p=History.htm

To do a complete search, I will have to use a college library which is usually not green-walled. Has anyone done any work with acenaphthene (http://en.wikipedia.org/wiki/Acenaphthene) or elevated temperatures?

Mike
Zen_Man
Ottawa, KS
(Zone 5b)

January 2, 2011
11:41 AM

Post #8288377

Mike,

"...1g of colchicine will cost $126.25 plus standard shipping rates..."

A gram of colchicine could treat a large number of plants, because you dilute it a lot to produce the solution that is applied to the growing points of plants to double their chromosomes. And you could apply very little of the diluted solution. Properly stored, a gram of colchicine could be enough to last for many years of amateur plant breeding.

Thanks for the info about the recent FDA ruling on the use of colchicine as a pharmaceutical. Considering the rigorous precautions that one is advised to take in using colchicine in plant breeding, it is almost humorous that some people are popping colchicine pills.

Since my primary interest is in zinnias, I will do some research to see if there are any effective alternatives to colchicine for doubling the chromosomes of zinnias. I am going to be looking for information much more recent than 1938. Hopefully work that has been done in the last 20 years.

ZM

This message was edited Jan 2, 2011 1:51 PM
dany12
szarvas
Hungary

January 3, 2011
1:49 AM

Post #8289468

An idea !
If nicotine work, skin patch with nicotine are working too ?
Michael_Ronayne
Nutley, NJ
(Zone 6b)

January 3, 2011
6:12 AM

Post #8289696

ZM,

For now I am going to focus on the thirty year period from 1931 to 1960 to see if there were other chemicals which were almost as good as Colchicine but didnít become popular. For now Acenaphthene and elevated temperatures are areas of interest. Another question is, will the use of plant hormones to accelerate root or leaf development enhance the chances of success using Polyploidy inducing chemicals. Note that I have found posts indicating that they should not be used.

A year ago the DG member Starlight1153 suggested that I search the Daylily Forum and I found this chemical supply company which may be useful as they have offices in three states including New Jersey.

Acenaphthene
http://www.advance-scientific.com/Properties.asp?Catalog_No=A2050
Colchicine, USP
http://www.advance-scientific.com/Properties.asp?Catalog_No=CO115

The other bit of information which I found was that Daylily breeders were obtaining prescriptions for generic Colchicine and dissolving the tablets to treat their Daylilyís. This will now get you, your doctor and your pharmacist in serious difficulties, not to mention that the price has most likely increased several orders of magnitude. For those of us who donít have easy access to a research lab, Daylily conversion using Colchicine may be a thing of the past.

In going through the Daylily Forum I also found some links, including ones which Starlight1153 had previously referenced.

PDS Colchicine Conversion Clinic by George Doorakian (With pictures)
http://patriotdaylilysociety.org/colchicine05.htm

Tetraploidy Conversion
http://members.tripod.com/~h_syriacus/tetraploidy.htm

Use this Google Search to find additional useful information on Daylily conversions.
http://www.google.com/#q=daylily+conversion

If my 2011 breeding plans work out, I will have Hibiscus moscheutos x Hibiscus mutabilis hybrids which will be sterile unless I can double the chromosome numbers. I will need a solution in the next nine months.

Mike
Michael_Ronayne
Nutley, NJ
(Zone 6b)

January 3, 2011
6:20 AM

Post #8289711

Dany12,

It is not clear how well Nicotine will work on any given plant species and assuming that it did work, extracting it from a medical patch would most likely be an expensive and difficult process. Extracting Nicotine from Tobacco leaf only requires boiling water.

Mike
Zen_Man
Ottawa, KS
(Zone 5b)

January 3, 2011
8:22 AM

Post #8289962

Mike,

"If my 2011 breeding plans work out, I will have Hibiscus moscheutos x Hibiscus mutabilis hybrids which will be sterile unless I can double the chromosome numbers. I will need a solution in the next nine months."

Another approach would be to propagate your sterile hybrids asexually, by cuttings or by micropropagation, using Tissue Culture.

http://www.kitchenculturekit.com/index.htm

I purchased one of their "kitchen culture kits" and I have been dabbling in tissue culture, as a means of multiplying any unique hybrid zinnia specimens that might occur for me. I have learned to grow zinnias from cuttings, but Tissue Culture offers the possibility of a much higher multiplication factor via micropropagation. That would in turn offer a much larger seed yield from a unique specimen to jumpstart the dehybridization process.

Besides micropropagation, (which is essentially propagation by the multiplication of very small cuttings), Tissue Culture offers other creative possibilities, like embryo rescue, somatic hybridization (cybridization), and pollen/anther culture to create haploid plants.

ZM
(not associated with any product or vendor mentioned)
Michael_Ronayne
Nutley, NJ
(Zone 6b)

January 3, 2011
11:34 AM

Post #8290241

ZM,

I am looking to produce a hardy hybrid Hibiscus with extra petals and to do that cuttings or tissue culture will not work. My success rate on cuttings is over 50% with most Hibiscus and also 100% for H. mutabilis, which anyone can root. There are sterile Hibiscus moscheutos x Hibiscus mutabilis hybrids on the market but they are not identified as such. What I need to do is get a second generation from the hybrid.

The second route to a multi petal hardy Hibiscus is the ďAnnie J. HemmingĒ (PP835) which will produce sever petal flowers. The Hemming family is still propagating this Hibiscus which was issued the first Hibiscus patented in 1949. The Hemming family has verified that the Hibiscus is still producing seven petal flowers and it is suspected that we are dealing with a chimera, for which tissue culture may be required to isolate the desired cells. We will try cuttings and seeds from the seven petal flowers first.

Mike
Michael_Ronayne
Nutley, NJ
(Zone 6b)

January 4, 2011
5:39 AM

Post #8291579

I just located a 2009 paper reporting the successful conversion of Hibiscus using Oryzalin, which reports that Oryzalin, is more effective than Colchicine. I have the PDF of the paper which makes very interesting reading. If the techniques described in the paper can be applied to other Hibiscus species, this means that we can convert Hibiscus without using Colchicine.

Ryan's Home Page
http://hort.oregonstate.edu/faculty-staff/contreras

ē Contreras, R.N., J.M. Ruter, and W.W. Hanna. 2009. An Oryzalin-Induced Autoallooctoploid of Hibiscus acetosella Welw. ex Hiern. ĎPanama Redí (Malvaceae). J. Amer. Soc. Hort. Sci. 134(5):553-559.

ē Contreras, Ryan N. and John M. Ruter. 2009. An oryzalin induced polyploidy from a hybrid of Hibiscus acetosella x H. radiatus (Malvaceae) exhibits reduced fertility and altered morphology. Proc. 106th Ann. Amer. Soc. Hort. Sci. Res. Conf. Hort. Science 44(4):1177.

An Oryzalin Induced Polyploid From a Hybrid of Hibiscus Acetosella x H. Radiatus (Malvaceae) Exhibits Reduced Fertility and Altered Morphology, Poster Board #305
http://ashs.confex.com/ashs/2009/webprogram/Paper2029.html

Inexpensive Oryzalin is available on eBay in small quantities with reasonable shipping costs from several sellers under the brand name ďSURFLAN ASĒ.

SURFLAN AS Pre-Emergent Herbicide 40.4% Oryzalin 8oz!
http://cgi.ebay.com/ws/eBayISAPI.dll?ViewItem&item=370356969508

http://cgi.ebay.com/ws/eBayISAPI.dll?ViewItem&item=120660433260
SURFLAN AS PreEmergent Herbicide 40.4% Oryzalin 8 oz

Mike
Dave67
New Port Richey, FL
(Zone 9b)

January 4, 2011
1:11 PM

Post #8292260

I found the methods used in this research paper interesting...
http://www.ces.ncsu.edu/fletcher/staff/tranney/polyploid.pdf

Dave

This message was edited Jan 9, 2011 6:52 PM
Dave67
New Port Richey, FL
(Zone 9b)

January 4, 2011
4:06 PM

Post #8292513

Link for making a working solution, using pure Oryzalin and SURFLAN AS
http://plant-tc.cfans.umn.edu/listserv/1999/log9908/msg00234.html



RickCorey_WA

RickCorey_WA
Everett, WA
(Zone 8a)

January 6, 2011
12:13 PM

Post #8295608

I would like to reassure anyone afraid of working with dangerous chemicals that it can be safe if you are always careful. To be sure your technique is safe, consider doing the proceedure first with a slurry of cayenne pepper and water soluble ink (or acid used for etching concrete). If your eyes, tongue and skin aren't burning or stained, you were careful enough.

Colchicine is considered "very toxic" and can be absorbed through the skin, and some effects are cumulative, but it ain't Plutonium or cyanide.

The LD50 in mice is a big 6 mg/kg, so you might have to eat 1/4 or 1/2 a GRAM to actually die promptly. I'm sure it would take much less to raise a blister or damage your cornea, but clearly we aren't talking micrograms, we're talking tens of milligrams. And sounds like a burning irritant, so you would know if you were being exposed.

So use gloves. If at any point dust or spray might blow around, use a filter mask and shower and wash your hair afterwards.

I used to work with radio-isotopes, tumor viruses and sterile tissue culture, then dusty Class 4 carcinogens, volatile toxic organic chemicals, caustics and strong acids including concentrated nitric acid. Just be careful. (But I did turn down a job with New England Nuclear, where I would have had to work with a CURIE per day! Not for me, thank you.)

However, it is very easy to forget simple things!

Like, "dusts and sprays blow around as you inhale and exhale".
"One dirty thing contaminates everything it touches, even if it doesn't LOOK dirty".
"Your gloves are dirty, and so are anything they touch."
"It is very easy to knock a bottle over so its contents run down your pants."

If you had to worry about micrograms, instead of tens of milligrams, the following would be usefull reminders:

It's easy to relax when you're "done", but I think the greatest risk comes during cleanup and as you pull off your gloves.

First clean up everything.
Then THROW AWAY away all rags and paper used during cleanup.
(Then maybe clean up again where your rags were.)
Pull off your gloves by turning them inside-out and discarding.
(Or wash them twice while wearing them, if they are not disposable.
After the palms and fingers are clean, consider washing the cuffs again.)
After pulling the gloves off and putting them where no one else will use them, wash your hands, wrists and lower arms.
THEN you can touch your eyes, mouth and face.

If it takes a whole milligram of something to hurt you, ignore all this and just keep it off your hands and out of your lungs. Don't leave any laying around. Lock the bottle where no one will mistake it for a spice. Don't knock it over or let the label fade or fall off.

While grinding some Thai chili peppers, I was proud of my technique until, about a day later, I relaized that after using a napkin to do initial wipe-downs, I foolishly laid the dirty napkin down "somewhere" as I wiped other things. For weeks, my kitchen was like a bio-hazard minefield where my hands would touch "something", and then later my lips or eyes would burn.

DUHH'NT you do that!

Corey
Michael_Ronayne
Nutley, NJ
(Zone 6b)

January 10, 2011
3:32 PM

Post #8303148

Corey,

If memory services my correctly I believe the specific treatment for Plutonium contamination is amputation where that is possible. If Oryzalin works it is a much safer alternative to Colchicine.

Mike

RickCorey_WA

RickCorey_WA
Everett, WA
(Zone 8a)

January 10, 2011
4:05 PM

Post #8303230

The phrase I recall is "immediate high amputation".

from the symptoms I've read about, even if the contamination was in your head, amputation might be preferable for waiting for it to work its way throguh your system.

When I worked at a chemical plant, they said that when there was a cyanide leak, ahnd you saw someone "go to one knee", DON'T try to help him. Just run.

Phosgene, on the other hand, is almost friendly. It smells like freshly-mown grass, and I think that if you only get a little, they can still save some of your lungs and hence it IS practical to try to drag someone out even if he is down and thrashing around.

Colchicine, Oryzalin, bah. It would probably take so much to kill you that you could SEE it! And its always 5reassuring to har things like "burning, blistering pain". That way, you KNOW if you are being exposed.

User-friendly.

;-)

Corey
SouthernIndiana
Underwood, IN

December 1, 2011
10:48 AM

Post #8912576

Does anyone have any new information about Colchicine, and where you can get it? And what forms that are available? Powder form, tablet, liquid? Thanks
IndaShade
Kylertown, PA
(Zone 5b)

December 1, 2011
12:51 PM

Post #8912722

If you are on good terms with your doctor, you can get a scrip for colchicine tablets.
Zen_Man
Ottawa, KS
(Zone 5b)

December 1, 2011
4:24 PM

Post #8912963

SouthernIndiana,

"Does anyone have any new information about Colchicine, and where you can get it?"

It's not new information, but back on January 3, 2011 at 08:12 AM, Michael_Ronayne provided a link to a Colchicine listing from Advance Scientific & Chemical. Their smaller amounts listed were 250mg for $49.64 or 1 gram for $115.61. They described it as a solid. I don't know what restrictions might apply to its sale. I guess you could find out by trying to buy some.

ZM

irisMA

irisMA
South Hamilton, MA

December 1, 2011
7:54 PM

Post #8913248

Not something to use if you don't know how.
IndaShade
Kylertown, PA
(Zone 5b)

December 3, 2011
6:07 AM

Post #8914657

You definitely have to respect the stuff. I wear gloves, googles, and a long sleeve shirt when I work with it, and do it over the sink. I have been treating individual phylloclades of Easter and Christmas cactus to see if i can get anything funky. I originally got it to treat diploid daylilies, but i only have one or two of those now and never got around to it.
Zen_Man
Ottawa, KS
(Zone 5b)

December 3, 2011
7:15 AM

Post #8914710

IndaShade,

You might be able to get some mutations via "somaclonal variation" by using Tissue Culture. A few possible links:

http://www.globalsciencebooks.info/JournalsSup/07FOB_1_1.html

http://www.springerlink.com/content/l62l1388437x0147/

http://www.springerlink.com/content/tw06753325564794/

http://www.jstor.org/pss/4293055

http://www.lapshin.org/cultivar/N36/pogany-l.htm

And, if you enjoy drinking from the Google fire hydrant, you could do a Google search using

cactus "somaclonal variation" easter OR christmas

I tried embedding that link here, but the forum somehow mangled it to just "cactus"

Some people who are propagating via Tissue Culture regard somaclonal variation as an annoyance, but it is also a possible source of new cultivars.

In vitro mutagenesis deliberately induces mutations via the use of chemical mutagens in tissue cultures. The chemical mutagens are all highly toxic and potentially carcinogenic, so they should be used with due precautions, including face masks and protective clothing. If skin contact occurs, the area should be thoroughly washed without delay.

ZM


This message was edited Dec 3, 2011 9:21 AM

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