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Author Topic: Colchicine experiment  (Read 9385 times)

Ian Morris

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Colchicine experiment
« on: January 30, 2015, 08:13:49 pm »

I have a reliable and constant source for colchicine and because of the incredible effects it may have on plant genetics I was wanting to do an experiment.

Before the project gets too far along I have some questions for the community here;

1) Has anyone had any first hand experience with colchicine and plant genetics?

2) Any theoretical/actual methods of administration?

3) Suggested plants for experimentation?

4) Any bio-ethical considerations I might be forgetting?

My thoughts;

5) My intention was to run multiple simultaneous experiments.  I would like to do a control with each experiment and would ultimately like to add colchicine in the seed stage for one experiment and then on perhaps the same species later on to see the total effects from embryo onward.  I was thinking T. Peruvian for this simply because it is probably the only plant I have in large enough supply but my hesitation would be that Trich seeds are not likely, even Peruvian seeds, to be exactly close genetically and the experiment would be really shaky.

Ill add more and obviously do a grow log but the purpose of this post is to brain storm ides for experimentation.

-Ian


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XDX

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Re: Colchicine experiment
« Reply #1 on: January 30, 2015, 09:17:58 pm »

I am totally unfamiliar with this Chem, this is the first time ever heard of it, so I've spent a whole 10 mins researching....

Can you expand a lil on ur hypothesis as to what ur expected result may be by doing colchicine treatment?
From the limited reading I just did, I would think that this may have use with genetically sterile plants... The two that come to mind are salvia divinorum and methysticodendron amesianum (aka Brugmansia culebra).
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Auxin

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Re: Colchicine experiment
« Reply #2 on: January 30, 2015, 09:32:17 pm »

For each species there is a species-specific minimum effective concentration of colchicine for polyploid induction. In plants that minimum concentration is usually somewhere below 0.01%.
In seed treatment it is typically used somewhere in the range of 0.01-0.1%.
If a concentration too high above the minimum effective threshold is used, the treated plants can be stunted and lack vigor. For instance in a clover species that was made polyploid 0.01% worked better than 0.025%, both having been treated for 8 hours. However, some researchers find that going overboard on concentration- while it kills many and stunts the survivors- can be more effective in making the survivors polyploid.
The fewer cells, the better. If you treat one cell, the resultant effect will be homogenous throughout the plant. If you treat a clump of cells, particularly ones in various stages of division, you'll get a 'mixoploid'- a sort of chimera. Because of this, many people treat seeds. However, many researchers had quite good success by treating seeds just as they germinated.
Colchicine is rapidly destroyed by sunlight.
Some survivors will be diploid.
In early work, staining and counting chromosomes was a bitch. As such, and because some treated plants will be diploid, researchers hoped that cell size was altered. By doubling or quadrupling the nuclear DNA compliment, the cell nucleus is enlarged. This often (but not always) leads to enlarged whole cells. If researchers identified variance in size on the treated plants versus controls they would test all treated plants first by staining and measuring cells in pollen or a leaf of a given size, then they'd do the chromosome count tests on subjects with enlarged cells. Comparing pollen cell size at home would be significantly easier than counting chromosomes at home.
If your target species is self-incompatible, grow out as many apparent successes as you can to increase chances of identifying successful breeding pairs.

@XDX Polyploidy is often used to make bigger, prettier, or more vigorous plants. Most ornamental roses and many morning glories you see are polyploids. Its also how things like seedless watermelons and grapes are made (they are triploids fertilized by diploids).
There are other uses too, including potential in improving low-genetic diversity things like S. divinorum or saffron.
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Ian Morris

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Re: Colchicine experiment
« Reply #3 on: January 30, 2015, 11:29:07 pm »

Wow thanks Auxin, im searching scholarly articles now.

Im guessing based on your handle and interests, that you deal professionally with plant genetics?   ;) rhetorical, don't feel compelled to answer if you wish to remain anonymous, I just appreciate the expert information.

Thanks guys,
Ian
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Sunshine

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Re: Colchicine experiment
« Reply #4 on: January 31, 2015, 12:45:13 am »

I've never dealt with or heard of colchicine. I know that a lot of these plant mutagen chemicals have carcinogenic risks associated with them. Do you have any idea of it's toxicity? I'd be interested in playing around with some on a few salvia cuttings if it's non-toxic to humans.

I've only played with a couple plant growth hormones such as 6-benzylamylpurine(6-BAP) and run of the mill rooting hormone. I imagine like 6-BAP you'd have to dissolve it in water or alcohol and apply it as a spray. I don't see any bio-ethical problems with altering a plant chemically. I'd err on the side of caution and isolate any plant you're experimenting on though. The only problems I can see coming about is creating a potentially invasive variation of the plant you're treating, unless you're treating a plant which you plan on consuming. Messing about with genetics can be dangerous. Just think of the effect some genetically modified foods have. There's a reason we hate Monsanto here. ;)

I can't wait to see what you come up with man. Keep us posted. :)
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Auxin

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Re: Colchicine experiment
« Reply #5 on: January 31, 2015, 02:41:55 am »

Its a deadly poison in humans, lol
It has long been used as a treatment for gout (a disease where people eat too much meat and crystals grow in their joints) but, despite being more effective, its only the emergency backup today because as little as 7 mg has been fatal and 65 mg is eastimated as the dose that would kill half of adults. Seven mg isnt much. Dont use it as a spray. One drop of a 1% solution has roughly 0.5 mg.
Its used primarily as a soak or as a paste/goop. Soak for seeds, seedlings, tissue culture blobs. Paste/goop for orchid buds, tree branch buds, etc. (often as a lanolin based paste but I've seen vegan versions in the literature as far back as the 30's)
It doesnt seem to be carcinogenic because rather than changing the code of DNA it just messes around whole chromosomes. So just dont dangle your fun bits in it before breeding.

[No, I'm not a professional. I'm just a psychedelic hippie nerd that reads a lot ;) ]
« Last Edit: January 31, 2015, 02:44:38 am by Auxin »
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Sunshine

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Re: Colchicine experiment
« Reply #6 on: January 31, 2015, 11:41:00 am »

Jeez, that's strong stuff! I'm not surprised. I know that nicotine has a LD50 of around 50-100mg for an average sized adult male. That'd make colchicine about the same strength as 10% nicotine solution in terms of toxicity. I'm not sure about nicotine's LLD. It sounds potent as hell, but yet still within workable range.

Are there any chemicals which can be used to render colchicine inactive? If you're doing this at home it'd be nice to be able to have some type of spray on hand to that you can use to decontaminate everything you've touched afterwards so you wouldn't have to worry about poisoning yourself and others.

You should defiantly consider taking proper safety precautions, Ian. I hope your source is shipping it to you pre-cut and not pure.  :o
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gnosis

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Re: Colchicine experiment
« Reply #7 on: January 31, 2015, 03:06:36 pm »

OK so you guys are saying this is used to develop your non seed bearing plants into seed bearing plants?

If so then I would love to try this on my Psychotria Nexus plants.  Can someone hook a brother up?
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BubbleCat

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Re: Colchicine experiment
« Reply #8 on: January 31, 2015, 05:05:43 pm »

I can tell it has worked on a Meconopsis ssp. it will not only "change infertile / sterile to fertile ones" but generally create multiploid genetics. I think its the "Lingsholm" Meconopsis that is a hybrid that sets seed, if Im not mistaking.
Edit: I do know size has sometimes been archieved with col. too
« Last Edit: January 31, 2015, 05:20:45 pm by BubbleCat »
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ONandONandON

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Re: Colchicine experiment
« Reply #9 on: February 08, 2015, 09:09:42 pm »

i could be wrong, but think i read polyploid plants are females,
so infertile unless you grow a regular male plant to pollinate.
Either way, it would be interesting to use Salvia Divinorum.
 8)
i have a similar experiment using Oryzalin specialty herbicide.
Just planted seeds that were soaked in Oryzaklin0.05%+H20.
That's another post though. Be careful please, and GOOD+luck!
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BubbleCat

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Re: Colchicine experiment
« Reply #10 on: February 08, 2015, 10:17:20 pm »

A Celosia Argentea does have 12 sets of chroms and they arent all female ;)

12n = 108 (dodecaploid)
« Last Edit: February 08, 2015, 10:28:44 pm by BubbleCat »
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misplant

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Re: Colchicine experiment
« Reply #11 on: December 22, 2015, 06:42:11 am »

I experimented with this in the late 70's, soaking some mj seeds in the juices from a crushed flower bulb.   One result observed in two plants were variegated leaves, another plant went triploid as a seedling and grew into a christmas tree shaped plant with nodes every inch or so up the stalk.
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chilito

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Re: Colchicine experiment
« Reply #12 on: December 22, 2015, 03:12:25 pm »

Ian morris,
Here is a good website( http://members.tripod.com/~h_syriacus/tetraploidy.htm ) that show methods for both colchicine and oryzalin tetraploid conversions for daylilies, from what I have learned in school oryzalin is much safer to use and has a better rate of conversion. I am sure you can rework this guys methods for what ever type of plant you are going to work with. Also some professor at Oregon state has a blog about oryzalin tetraploid conversions, I will try and find a link to his work and post it soon.

Edited to remove mention of certain plant.
« Last Edit: December 23, 2015, 08:31:32 pm by chilito »
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bezevo

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Re: Colchicine experiment
« Reply #13 on: June 19, 2016, 12:20:11 am »

Ian  have you started  treating any  T.peruvianus  seeds or sprots yet /tis is  very interesting 
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ONandONandON

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Re: Colchicine experiment
« Reply #14 on: February 23, 2019, 09:08:58 pm »

Quote
Experiment 5: Anther Culture
 
Background
 
 The purpose of anther and pollen culture is the production of haploid plants by the induction of embryogenesis from repeated divisions of microspores or immature pollen grains (Dodds and Roberts, 1985).  The chromosome complement of these haploids can be doubled by colchicine treatment or other techniques to yield fertile homozygous diploids.  The resultant diploids can be used in plant breeding to improve crop plants (Sunderland and Cocking, 1978).
168    Plant Tissue Culture
 
 The haploid nature of embryoids should be determined by standard chromosome staining procedures (acetocarmine or Feulgen reaction) prior to colchicine treatment.  Similarly, the effect of colchicine on chromosome doubling needs to be monitored by chromosome staining.
 
Materials
 
 tobacco buds  sharp pointed forceps, surgical scissors  95% ethanol  petri dishes of culture medium (1/2 strength MS, 2% sucrose 0.8% agar, glutamine [800 mg/liter], serine [100 mg/liter])
 
 
Methods 
 
1. Obtain two buds at the appropriate stage.  This occurs in tobacco when the sepals and the petals in the bud are the same length.
 
2. Holding the bud by the pedicel between the thumb and first finger, dip the entire bud in 95% ethanol for 15 seconds
 
3. Remove bud and allow excess alcohol to drip off.
 
4. With a pair of sterile forceps, remove the outer layer of tissue, the sepals.
 
5. Next, remove the inner layer of tissue, the petals, exposing the anthers.
 
6. Open the petri dish containing the medium for the induction of haploids.  Remove each anther from the bud and drop it onto the medium.  Do not damage the anther or include any filament tissue.
 
7. Repeat for another bud.
 
8. When finished, seal the plates and place in incubator (25°C).
 
9. In 2–3 weeks examine for somatic embryo initiation.  Embryoid-forming cells are characterized by dense cytoplasmic contents, large starch grains and a relatively large nucleus.  Embryoids appear opaque among translucent cells.  Embryoids also exhibit high dehydrogenase activity and can be detected by tetrazolium staining (Dodds and Roberts, 1985).
 
Acknowledgements
 
 Paul Bottino (Botany Department, University of Maryland, College Park) provided the sterilization procedure for Experiment 2 and Methods for Experiments 4 and 5.
 
 Plant Tissue Culture        169
 
 
Literature Cited
 
Bottino, P. J.  1981.  Methods in plant tissue culture.  Kemtec Educational Corp., Kensington, Maryland, 72 pages. Butcher, D. N., and D. S. Ingram.  1976.  Plant tissue culture.  Arnold, London, 67 pages. Dodds, J. H., and L. W. Roberts.  1985.  Experiments in plant tissue culture.  Second edition.  Cambridge University Press, New York, 232 pages. Street, H. E.  1973.  Plant tissue and cell culture.  Blackwell Scientific Publications, Oxford, 320 pages. Sunderland, N., and E. C. Cocking.  1978.  Plant tissue culture in China—major change ahead?  Nature, 274:643-44. Sunderland, N., and M. Roberts.  1977.  New approach to pollen culture.  Nature, 270:236-238. Ting, I. P.  1982.  Plant physiology.  Addison-Wesley, Reading, Massachusetts, 642 pages. Wetherell, D. F.  1982.  Introduction to "in vitro" propagation.  Avery Publishing Group Inc., Wayne
« Last Edit: February 23, 2019, 09:09:23 pm by ONandONandON »
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