49 replies to this topic

[coorsmikey]

This thread’s purpose is to gather as much information as possible into one place for reference and to discuss the history of how colchicum has been used medicinally, in botany and mycology. It will be a place where I can store citing and info collected as well as what other have to add. Share my work and progress with the input from others that are familiar with the many beneficial uses these alkaloids have with many of the likeminded hobbies we read about here on Mycotopia. Also, a place for others to share their knowledge and experience.

Everything is for informational purposes only and I would not suggest to anyone to refine their own colchicine because of FDA regulations giving ULR Pharma exclusive rights to the Brand Colcrys, Colchicine is a regulated drug in the US. It is illegal to possess without a prescription and a federal offense to use in any other way that it is intended to use. However, most of the information available to the public is specific to Colchicine even though we are discussing naturally occurring alkaloids from colchicum Autumnale and some other species of plant that contain the same alkaloids.and all information to be contained here is for reference. Also arguably there are risks associated that I want to be addressed here with documentation. These alkaloids ARE toxic and hormesis. Do not attempt to repeat the result of information found in this now or in the future with the proper organic chemistry knowledge and education. The abstracts the will be added here are for informational learning only.

I am just wanting a one place resource to store my finding and share my work. Also to bring awareness to the uses of colchicum and for Botanical and Mycology uses. I have personal experience with hybrid “Dikariotic” fungi strains with very successful and mixed results which I want to study more and release the information I have collected.

Please stay on topic and contribute what you have, know and find. I am especially calling out Microbe here to add his knowledge and experience

I will be adding and editing stuff as time permits. A lot of studies information is in science journals over the last hundred year. Many good papers that cost me money and have to acquire permission before can post them.

This will be under construction for some time but will be edited into a clean abstract at some point. A placeholder per se. Please include a reference and sources to whatever anyone may add that involves specific information regarding said alkaloids and avoid adding hearsay. Be advised that hearsay comments are subject to removal at the OP’s request. All information is welcome if it can be proven not to be hearsay. If anyone has any issue or concerns please contact me or report it and I will be happy to address with you.

Edited by coorsmikey, 24 January 2019 - 10:42 PM.

[coorsmikey]

What is colchicine and how is it used?

Contributed by PAXCO:

Polyploidy (favorable traits in Cannabis) has not been shown to occur naturally in Cannabis; however, it may be induced artificially with colchicine treatments. Colchicine is a poisonous compound extracted from the roots of certain Colchicum species; it inhibits chromosome segregation to daughter cells and cell wall formation, resulting in larger than average daughter cells with multiple chromosome sets.

The studies of H. E. Warmke et al. (1942-1944) seem to indicate that colchicine raised drug levels in Cannabis. It is unfortunate that Warmke was unaware of the actual psychoactive ingredients of Cannabis and was therefore unable to extract THC. His crude acetone extract and archaic techniques of bioassay using killifish and small freshwater crustaceans are far from conclusive. He was, however, able to produce both triploid and tetraploid strains of Cannabis with up to twice the potency of dip- bid strains (in their ability to kill small aquatic organisms). The aim of his research was to "produce a strain of hemp with materially reduced marijuana content" and his results indicated that polyploidy raised the potency of Cannabis without any apparent increase in fiber quality or yield.

Warmke's work with polyploids shed light on the nature of sexual determination in Cannabis. He also illustrated that potency is genetically determined by creating a lower potency strain of hemp through selective breeding with low potency parents. More recent research by A. I. Zhatov (1979) with fiber Cannabis showed that some economically valuable traits such as fiber quantity may be improved through polyploidy. Polyploids require more water and are usually more sensitive to changes in environment. Vegetative growth cycles are extended by up to 30-40% in polyploids. An extended vegetative period could delay the flowering of polyploid drug strains and interfere with the formation of floral clusters.

It would be difficult to determine if cannabinoid levels had been raised by polyploidy if polyploid plants were not able to mature fully in the favorable part of the season when cannabinoid production is promoted by plentiful light and warm temperatures. Greenhouses and artificial lighting can be used to extend the season and test polyploid strains. The height of tetraploid (4n) Cannabis in these experiments often exceeded the height of the original diploid plants by 25-30%. Tetraploids were intensely colored, with dark green leaves and stems and a well developed gross phenotype. Increased height and vigorous growth, as a rule, vanish in subsequent generations. Tetraploid plants often revert back to the diploid condition, making it difficult to support tetraploid populations. Frequent tests are performed to determine if ploidy is changing.

Triploid (3n) strains were formed with great difficulty by crossing artificially created tetraploids (4n) with dipbids (2n). Triploids proved to be inferior to both diploids and tetraploids in many cases. De Pasquale et al. (1979) conducted experiments with Cannabis which was treated with 0.25% and 0.50% solutions of colchicine at the primary meristem seven days after generation. Treated plants were slightly taller and possessed slightly larger leaves than the controls, Anoma- lies in leaf growth occurred in 20% and 39%, respectively, of the surviving treated plants. In the first group (0.25%) cannabinoid levels were highest in the plants without anomalies, and in the second group (0.50%) cannabinoid levels were highest in plants with anomalies.

Overall, treated plants showed a 166-250% increase in THC with respect to controls and a decrease of CBD (30-33%) and CBN (39-65%). CBD (cannabidiol) and CBN (cannabinol) are cannabinoids involved in the biosynthesis and degradation of THC. THC levels in the control plants were very low (less than 1%). Possibly colchicine or the resulting polyploidy interferes with cannabinoid biogenesis to favor THC. In treated plants with deformed leaf lamina, 90% of the cells are tetraploid (4n 40) and 10% diploid (2n 20). In treated plants without deformed lamina a few cells are tetraploid and the remainder are triploid or diploid.

The transformation of diploid plants to the tetraploid level inevitably results in the formation of a few plants with an unbalanced set of chromosomes (2n + 1, 2n - 1, etc.). These plants are called aneuploids. Aneuploids are inferior to polyploids in every economic respect. Aneuploid Cannabis is characterized by extremely small seeds. The weight of 1,000 seeds ranges from 7 to 9 grams (1/4 to 1/3 ounce). Under natural conditions diploid plants do not have such small seeds and average 14-19 grams (1/2- 2/3 ounce) per 1,000 (Zhatov 1979). Once again, little emphasis has been placed on the relationship between flower or resin production and polyploidy. Further research to determine the effect of polyploidy on these and other economically valuable traits of Cannabis is needed.

Colchicine is sold by laboratory supply houses, and breeders have used it to induce polyploldy in Cannabis. However, colchicine is poisonous, so special care is exercised by the breeder in any use of it. Many clandestine cultivators have started polyploid strains with colchicine. Except for changes in leaf shape and phyllotaxy, no out- standing characteristics have developed in these strains and potency seems unaffected. However, none of the strains have been examined to determine if they are actually polyploid or if they were merely treated with colchicine to no effect.

Seed treatment is the most effective and safest way to apply colchicine. * In this way, the entire plant growing from a colchicine-treated seed could be polyploid and if any colchicine exists at the end of the growing season the amount would be infinitesimal. Colchicine is nearly always lethal to Cannabis seeds, and in the treatment there is a very fine line between polyploidy and death. In other words, if 100 viable seeds are treated with colchicine and 40 of them germinate it is unlikely that the treatment induced polyploidy in any of the survivors. On the other hand, if 1,000 viable treated seeds give rise to 3 seedlings, the chances are better that they are polyploid since the treatment killed all of the seeds but those three.

It is still necessary to determine if the offspring are actually polyploid by microscopic examination. The work of Menzel (1964) presents us with a crude map of the chromosomes of Cannabis, Chromosomes 2-6 and 9 are distinguished by the length of each arm. Chromosome 1 is distinguished by a large knob on one end and a dark chromomere 1 micron from the knob. Chromosome 7 is extremely short and dense, and chromosome 8 is assumed to be the sex chromosome. In the future, chromosome *The word "safest" is used here as a relative term.

Colchicine has received recent media attention as a dangerous poison and while these accounts are probably a bit too lurid, the real dangers of exposure to coichicine have not been fully researched. The possibility of bodily harm exists and this is multiplied when breeders inexperienced in handling toxins use colchicine. Seed treatment might be safer than spraying a grown plant but the safest method of all is to not use colchicine. mapping will enable us to picture the location of the genes influencing the phenotype of Cannabis. This will enable geneticists to determine and manipulate the important characteristics contained in the gene pool. For each trait the number of genes in control will be known, which chromosomes carry them, and where they are located along those chromosomes.

(Taken from 'Marijuana Botany',R.C.Clarke,CH.3)

[Microbe]

I have a buddy who said he will get me some Colcichine and even mentioned rattlesnake venom. Colcichine is the way to go IMO and much less dangerous but how freaking cool would it be to have a some darts and a blow gun and a 15 ml Vacutainer of diamond back venom...and then when the ex-wifes come in.......wait pretend I didn't say that last part.

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[coorsmikey]

I think it’s safe to say that Colchicine’s dangers are clearly blown out of proportion especially on the OMCs. It is toxic in quantity and will give shits like no other. I can vouch for that first hand and personally. I also take 1.2mg daily with no side effects. Colchicine is very effective treatment for what it is prescribed to me for. While working in the lab, proper safety and precautions should always be best practiced. In no way am I saying it’s not dangerous. But some of the overreactions I see on the OMC are a bit much, tyvek suits and stuff. The irony that it is a carcinogen that effectively treats several kinds of cancer. Colchicum is toxic and should be handled with care. But used correctly with minimal amount it is hormesis.
Hopefully in time, this thread will have all the documentation including accurate ways to handle safely. Also hope make the facts available for others to clear up misconceptions.
By all means, if someone doesn’t know what their doing with any chemical. Please don’t try this shit at home! Transparency to the risks, safety and dangers is important. If anyone wants to say how dangerous it can be please do so and back it with your source. After all this threads intentions is to as much information as possible into one place for reference.

Source Drugs.com

Carcinogenesis, Mutagenesis, Impairment of Fertility
Carcinogenesis

Carcinogenicity studies of Colchicine have not been conducted. Due to the potential for Colchicine to produce aneuploid cells (cells with an unequal number of chromosomes), Colchicine presents a theoretical increased risk of malignancy.

Mutagenesis

Published studies demonstrated that Colchicine was negative for mutagenicity in the bacterial reverse mutation assay. However, in vitro chromosomal aberration assays demonstrated the formation of micronuclei following Colchicine treatment. Because published studies demonstrated that Colchicine induces aneuploidy through the process of mitotic nondisjunction without structural DNA changes, Colchicine is not considered clastogenic, although micronuclei are formed

Toxicity

Colchicine can be toxic when ingested, inhaled, or absorbed in the eyes. Colchicine can cause a temporary clouding of the cornea and be absorbed into the body, causing systemic toxicity. Symptoms of colchicine overdose start 2 to 24 hours after the toxic dose has been ingested and include burning in the mouth and throat, fever, vomiting, diarrhea, and abdominal pain. This can cause hypovolemic shock due to extreme vascular damage and fluid loss through the gastrointestinal tract, which can be fatal. If the affected person does not recover, they may enter the multiple-system organ failure phase of colchicine overdose. This includes kidney damage, which causes low urine output and bloody urine; low white blood cell counts that can last for several days; anemia; muscular weakness; liver failure; hepatomegaly; bone marrow suppression; thrombocytopenia; and ascending paralysis leading to potentially fatal respiratory failure. Neurologic symptoms are also evident, including seizures, confusion, and delirium; children may experience hallucinations. Recovery may begin within six to eight days and begins with rebound leukocytosis and alopecia as organ functions return to normal.[14]

Long-term exposure to colchicine can lead to toxicity, particularly of the bone marrow, kidney, and nerves. Effects of long-term colchicine toxicity include agranulocytosis, thrombocytopenia, low white blood cell counts, aplastic anemia, alopecia, rash, purpura, vesicular dermatitis, kidney damage, anuria, peripheral neuropathy, and myopathy.[14]

No specific antidote for colchicine is known, but supportive care is used in cases of overdose. In the immediate period after an overdose, monitoring for gastrointestinal symptoms, cardiac dysrhythmias, and respiratory depression is appropriate.[14] Certain common inhibitors of CYP3A4 and/or P-gp, including grapefruit juice, may increase the risk of colchicine toxicity

Quote source Wiki

In 2012 Asia’s biggest drugmaker — Takeda Pharmaceutical Co. — acquired URL Pharma for $800 million including the rights to colchicine (brand name Colcrys) earning $1.2 billion in revenue by raising the price even more.[24]

Oral colchicine had been used for many years as an unapproved drug with no FDA-approved prescribing information, dosage recommendations, or drug interaction warnings.[25] On July 30, 2009 the FDA approved colchicine as a monotherapy for the treatment of three different indications (familial Mediterranean fever, acute gout flares, and for the prophylaxis of gout flares[25]), and gave URL Pharma a three-year marketing exclusivity agreement[26] in exchange for URL Pharma doing 17 new studies and investing $100 million into the product, of which $45 million went to the FDA for the application fee. URL Pharma raised the price from $0.09 per tablet to $4.85, and the FDA removed the older unapproved colchicine from the market in October 2010, both in oral and intravenous forms, but gave pharmacies the opportunity to buy up the older unapproved colchicine.[27] Colchicine in combination with probenecid has been FDA-approved prior to 1982.

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Edited by coorsmikey, 01 October 2017 - 04:23 PM.

[coorsmikey]

ARX, The results of colchicine and two dikaryotic substrains

All references to colchicine here are actually extracts of Colchicum Autumnale.

The variations of Ps. cubensis used for this cross or mutation were from cloned fruitbodies transferred to MEA to isolate strain variations and eliminate sectoring. One strain used was a 6yr old APE culture that had gradual deterioration of its mutated characteristics. This cultivar is said to be a cross of Albino PF and Penis Envy had lost the standard characteristics of short albino bowling pin looking fruits that did not produce any visible spores. It eventually began to resemble one of its parents, producing normal leucistic fruitbodies that produced spores. I suspect that it has basically reverted back to original Albino PF. Attempts were made to revive the APE hybrid phenotypes from dried specimens but still resulted in normal looking fruitbodies. Below pictured from left to right over years of cloning.

 

Senescence or degradation of APE clone over several years

The other strain was Redboy. Personally worked by me since 2007, this strain was very aggressive fast fruiter that never had any complaints from my friends that I shared with. Over the years of working this strain, it has developed certain recognizable characteristics like dimpled caps and striped stipes that you'll see carried over to the first and second generation of the hybrid.

    

Redboy var.

Clones were made from each strain and grown out on standard MEA. Note that these clones were mated dikaryotic mycelium. Transfers of each strain were made to the following. 60 ml of MEA prepared with 0.6 mg Colchicine added and poured between four ½ pint jars with approx 15 ml in each. Sterilized at 15 PSI for 15 minutes.

All had a transfer on each side of the agar and let to grow out until meeting each other. It was clear where the two strains meet like hitting a wall. Transfers of mycelium were taken from the intersecting points within minutes of the intersection. The cultures were then allowed to continue growing. To my surprise, all four cultures developed a third distinctly different sector of growth. The original transfer from the intersecting points was grown out and showed no signs of any mutations that weren’t there previously. They all appeared to be one or the other of the two strain used. Meanwhile further transfers were done from the mysterious third sector on all four cultures.

 

Third sector indicating mating of two dikaryotic substrain on agar containing colchicine

The transfer from the third sectors was grown out and exhibited different behaviors from the original strains except for one. Three of the four show exceptionally fast colonization and very aggressive pinsets. Two of these had pigmented pins while one appeared to be leucistic. These three would develop massive pinsets but the whole colony would just abort. They were cleaned and dunked just to repeat the same results on future flushes. They were eventually abandoned and no further research was done. The fourth appeared as it had not changed and resembled the Redboy strain. At this time I pretty much had thought I would have to start over and wrote off the experiment as a failure. I was convinced that the colchicine had worked but I just failed to have a successful cross. I ended up growing out the strain that I thought was essentially a Redboy clone and took several prints. I labeled these prints “Redboy X” so I could track the behaviors and prevent them from being mixed up with my regular print stock.

1st generation of successful mating                  Other matings that failed to fruit

After spores from the prints labeled Redboy X was grown out and fruited it became obvious that a successful cross or mutation had indeed happened. The second generation of this was exhibiting multiple phenotypes side by side. Phenotypes familiar from both of the original strains were fruit from the same substrate. With clusters of fruitbodies that contained both leucistic and pigmented mushrooms. This reminded me of early generations of F Albino created by TheChosenOne.

Not the best pics, but you can see the different phenotypes growing together

(photos left to right from SmokeyMountainGirl and PinkMenace)

Multiple spore prints from the second generation leucistic specimens were then grown out on standard MEA to fruition. The results from all the prints were consistent in which they produced albino fruitbodies that produced no visible spores. After seeing these consistent results I was fairly confident that the hybrid/ mutation was stable. Another print from the second generation leucistic fruits was used to inoculate All-in-one myco-bags. A substrate consisting of 4 cup wheat berries, 8 cup coir, and 4 cup BRF. 6 cup water to hydrate, Grain at the bottom of the bags, coir, and BRF mixed together on top of the grain. After sterilization, the grain at the bottom of the bags was inoculated with the spores then allowed to colonize at room temp. Once the majority of the grain was colonized, the grain mixed with the rest of the substrate.

MEA test results

The results from fruiting on a better substrate than agar were full-size mushrooms that are pure white. Mostly sporeless, there are a few random fruits out many that I was able to print or swab. These are Labeled ARX (F-4) and have been quartered to share with the OMC. The 4 generations have not been grown out to fruition yet but I suspect that the genes are stable and will produce the same phenotypes as F-3. I am curious to see if F-4 produces any spores at all. I have the feeling they will be sterile producing no spores. These spores have been released to the OMC and we should see the results soon.

ARX

Edited by coorsmikey, 25 January 2019 - 07:09 PM.

[Microbe]

UV light will not work for my application but I do appreciate the warnings but I have already researched it extensively and I have kitchen chemistry skills at best or at least the practical skills of a kitchen chem but no advance chemistry skills is required for handling colchicine.....it just needs to be handled as any any prescription medication would be such as childproof container, out of reach and etc.

I understand the risk of over exposure to the particles floating in the air bit even then them I would imply caution and not a level such as warning or danger. I plan to make a suspended concentrate anyway so won't be messing with raw powder very often.

Out of everything I have researched this is by far the most interesting to me and will eventually consume on my time dedicated to my work.

Thanks again for caring enough to warn us of potential dangers and like Coors said if your not familiar with it or done some research don't mess with it. But that rule can apply universally to just about anything substance, drug, chemical, plant, mushroom, cultivating, and etc.



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[JanSteen]

Suit yourselves guys. It might take a while before the cancer shows up, but don't come knocking at my door when that happens. Just because you don't notice anything now, doesn't mean this shit is screwing with your stem cells already. It's a mitosis inhibitor, which means that your cells stop dividing and lump together in the most important process that keeps your body intact and functional. This might not have an acute effect, but it might just be passed on to your future offspring or you might develop some serious cancers by the time you're retiring.

I've worked with this stuff in the lab, and there's a reason why interns weren't even allowed in the same room as the stuff (apart from company policy). Please do not forget that pharmacists know the interactions of chemicals and do extensive testing on administration routes. That just one company was up to par on those tests and got an FDA approval, means a lot. Why don't other companies get the same approval? I mean, they have the money and the resources and they know that they would sell a truckload more if they just lower the price a single cent compared to the competition. The colchicine might be incorporated in other molecules that are specifically designed to only enter specific cell types. But nobody seems to think about that. I know that pharmacists usually keep those ingredients a secret, to prevent people from copying their recipe. There's no way in saying if that's the case. Unless you have a full chemical report that is..
Also remember that chemotherapy is also used to treat cancer. Why don't you use X-rays? Ironic isn't it. Those kind of arguments are why people get hurt doing this stuff at home. Radiologists don't notice that they've not been wearing the led apron until it's too late. They can take something close to 2000 doses before something bad happens.

If you think that making and working with a suspension doesn't release aerosols, then it's safe to assume that you need to update your kitchen chemistry skills some more. What's your solvent? At what temperature does it evaporate? If you can smell it at room temperature, it's in the air already. Which means there's aerosols. Never underestimate gas and fluid dynamics.

It would make a good case study, so please, note down the first time you've worked with this stuff, and let us know when you get the doctors call with the bad news.
I wish I was kidding, I would love to be kidding right now. But I'm dead serious. You might laugh about this, and feel that I'm over reacting.. But I'm reading here that some guy wants to make a concentrated suspension of the stuff. That scares me. It's something that could be seen as a biochemicall weapon. As a matter of fact, a concentrated suspension of this stuff IS a biochemical weapon.

Anyway, I came here to warn you. I did that. The rest is up to yourselves. I'm pulling my hands off of this. I don't want anything to do with this and I want you guys to know that I do not support you in any way. I hope the moderators have that insight as well.

[coorsmikey]

The following are the species included under Colchicum.[18] Many species previously classified under Androcymbium, Bulbocodium and Merendera were synonymized under Colchicum based on molecular genetic evidence, Androcymbium is currently considered a separate genus.

Colchicum agrippinum (probably a hybrid of garden origin)
Colchicum alpinum DC. in J.B.A.M.de Lamarck & A.P.de Candolle
Colchicum androcymbioides (Valdés) K.Perss.
Colchicum antepense K.Perss.
Colchicum antilibanoticum Gomb.
Colchicum arenarium Waldst. & Kit.
Colchicum arenasii Fridl.
Colchicum asteranthum Vassiliades & K.M.Perss.
Colchicum atticum Spruner ex Tommas.
Colchicum autumnale L.
Colchicum balansae Planch.
Colchicum baytopiorum C.D.Brickell
Colchicum bivonae Guss.
Colchicum boissieri Orph.
Colchicum bulbocodium Ker Gawl.
Colchicum burttii Meikle
Colchicum chalcedonicum Azn.
Colchicum chimonanthum K.Perss.
Colchicum chlorobasis K.Perss.
Colchicum cilicicum (Boiss.) Dammer
Colchicum confusum K.Perss.
Colchicum corsicum Baker
Colchicum cretense Greuter
Colchicum crocifolium Boiss.
Colchicum cupanii Guss.
Colchicum davisii C.D.Brickell
Colchicum decaisnei Boiss.
Colchicum doerfleri Halácsy
Colchicum dolichantherum K.Perss.
Colchicum eichleri (Regel) K.Perss.
Colchicum euboeum (Boiss.) K.Perss.
Colchicum fasciculare (L.) R.Br.
Colchicum feinbruniae K.Perss.
Colchicum figlalii (Varol) Parolly & Eren
Colchicum filifolium (Cambess.) Stef.
Colchicum freynii Bornm.
Colchicum gonarei Camarda
Colchicum graecum K.Perss.
Colchicum greuteri (Gabrieljan) K.Perss.
Colchicum haynaldii Heuff.
Colchicum heldreichii K.Perss.
Colchicum hierosolymitanum Feinbrun
Colchicum hirsutum Stef.
Colchicum hungaricum Janka
Colchicum ignescens K.Perss.
Colchicum imperatoris-friderici Siehe ex K.Perss.
Colchicum inundatum K.Perss.
Colchicum kesselringii Regel
Colchicum kotschyi Boiss.
Colchicum kurdicum (Bornm.) Stef.
Colchicum laetum Steven
Colchicum lagotum K.Perss.
Colchicum leptanthum K.Perss.
Colchicum lingulatum Boiss. & Spruner in P.E.Boissier
Colchicum longifolium Castagne
Colchicum lusitanum Brot.
Colchicum luteum Baker
Colchicum macedonicum Kosanin
Colchicum macrophyllum B.L.Burtt
Colchicum manissadjianii (Azn.) K.Perss.
Colchicum micaceum K.Perss.
Colchicum micranthum Boiss.
Colchicum minutum K.Perss.
Colchicum mirzoevae (Gabrieljan) K.Perss.
Colchicum montanum L.
Colchicum multiflorum Brot.
Colchicum munzurense K.Perss.
Colchicum nanum K.Perss.
Colchicum neapolitanum (Ten.) Ten.
Colchicum parlatoris Orph.
Colchicum parnassicum Sart., Orph. & Heldr. in P.E.Boissier
Colchicum paschei K.Perss.
Colchicum peloponnesiacum Rech.f. & P.H.Davis
Colchicum persicum Baker
Colchicum polyphyllum Boiss. & Heldr. in P.E.Boissier
Colchicum pulchellum K.Perss.
Colchicum pusillum Sieber
Colchicum raddeanum (Regel) K.Perss.
Colchicum rausii K.Perss.
Colchicum ritchii R.Br.
Colchicum robustum (Bunge) Stef.
Colchicum sanguicolle K.Perss.
Colchicum schimperi Janka ex Stef.
Colchicum serpentinum Woronow ex Miscz.
Colchicum sfikasianum Kit Tan & Iatroú
Colchicum sieheanum Hausskn. ex Stef.
Colchicum soboliferum (C.A.Mey.) Stef.
Colchicum speciosum Steven
Colchicum stevenii Kunth
Colchicum szovitsii Fisch. & C.A.Mey.
Colchicum trigynum (Steven ex Adam) Stearn
Colchicum triphyllum Kunze
Colchicum troodi Kotschy in F.Unger & C.G.T.Kotschy
Colchicum tunicatum Feinbrun
Colchicum turcicum Janka
Colchicum tuviae Feinbrun
Colchicum umbrosum Steven
Colchicum varians (Freyn & Bornm.) Dyer in B.D.Jackson
Colchicum variegatum L.
Colchicum wendelboi K.Perss.
Colchicum woronowii Bokeriya
Colchicum zahnii Heldr
source Wikipedia

[coorsmikey]

https://www.ncbi.nlm...les/PMC4202654/
 

Improved Growth and Colchicine Concentration in Gloriosa Superba on Mycorrhizal Inoculation Supplemented with Phosphorus-Fertilizer

Devendra Kumar Pandey,1 Tabarak Malik,1 Abhijit Dey,2 Joginder Singh,1 and RM Banik3


Author information Copyright and License information Disclaimer
 


 
 

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Abstract



Background
Gloriosa superba produces an array of alkaloids including colchicine, a compound of interest in the treatment of various diseases. The tuber of Gloriosa superba is a rich source of colchicine which has shown anti-gout, anti-inflammatory, and anti-tumor activity. However, this promising compound remains expensive and Gloriosa superba is such a good source in global scale. Increase in yield of naturally occurring colchicine is an important area of investigation.



Materials and Methods
The effects of inoculation by four arbuscular mycorrhizal (AM), fungi, Glomus mossae, Glomus fasciculatum, Gigaspora margarita and Gigaspora gilmorei either alone or supplemented with P-fertilizer, on colchicine concentration in Gloriosa superba were studied. The concentration of colchicine was determined by high-performance thin layer chromatography.



Results
The four fungi significantly increased concentration of colchicine in the herb. Although there was significant increase in concentration of colchicine in non-mycorrhizal P-fertilized plants as compared to control, the extent of the increase was less compared to mycorrhizal plants grown with or without P-fertilization. This suggests that the increase in colchicine concentration may not be entirely attributed to enhanced P-nutrition and improved growth. Among the four AM fungi Glomus mossae was found to be best. The total colchicine content of plant (mg / plant) was significantly high in plants inoculated with Glomus mossae and 25 mg kg−1phosphorus fertilizer (348.9 mg /plant) while the control contain least colchicine (177.87 mg / plant).



Conclusion
The study suggests a potential role of AM fungi in improving the concentration of colchicine in Gloriosa superba tuber.


Keywords: Gloriosa superba, colchicine, Arbuscular mycorrhiza, Phosphorus fertilizer

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Introduction
Gloriosa superba L. (Liliaceae) is an ornamental climbing herb native of tropical Asia, and Africa often been cultivated for its beautiful flowers. The roots and tubers of this plant have been used in traditional Indian medicine for the treatment of gout, rheumatic arthritis, diseases of the skin and liver, and several other purposes (Finnie and Staden, 1994). Since the detection of colchicine in Gloriosa (Clewer et al, 1915), a number of researchers have suggested that Gloriosa could serve as a commercial source of colchicine (Sarin et al, 1977; Srivastava and Chandra, 1977), as the colchicine content in the genera Colchicum has been reported to be lower than in Gloriosa (Bellet and Graignault, 1985). Colchicine, the main alkaloid of Gloriosa superba, was a useful agent in the treatment of acute attacks of gout (Robert et al., 1987) cirrhosis of the liver (Kershowtrich et al, 1988) and familial Mediterranean fever (Goldfinger et al. 1972). Colchicine and its analogues were used clinically for the treatment of certain forms of leukemia and solid tumers (Alexander et al, 1994). Due to its potent affinity for tubulin; colchicine is used in biological and breeding studies to produce polyploidy, multiplication of the chromosomes in cell nucleus and in tubulin binding assays as a positive control (Trease and Evans, 1983).
Since the discovery of colchicine in Gloriosa, its commercial importance has increased as it has a higher content of colchicine than Colchicum (Yoschida, 1988a; Finnie & van Staden 1994). In horticultural practice, vegetative propagation of Gloriosa is commonly used but the growth is very low (Kranse 1986). There are limited numbers of reports on tissue culture of G. superba (Finnie and van Staden, 1989). The amount of colchicine recorded in tissue cultures of Gloriosa was 10–25 times lower than those found in plants growing in vivo (Hayashi et al. 1988, Finnie & van Staden 1991). Therefore, improvement in naturally occurring colchicine yield is an important area of investigation.
Arbuscular mycorrhizal (AM) fungi are known to play a pivotal role in the nutrition and growth of plants in many production-orientated agricultural systems, but little is known about their potential effect on secondary metabolites in medicinal and aromatic plants (Copetta et al. 2006; Kapoor et al. 2002a, b, 2004; Khaosaad et al. 2006, Sailo and Bagyaraj, 2005). Gloriosa superba is usually both mycorrhizal and responsive to the symbiosis (Blanke et al. 2005). To our knowledge, no study has been carried out on the effect of AM in the production of colchicine in Gloriosa superba. We therefore conducted an experiment with the objectives to (1) compare the effects of four AM fungi Glomus mossae, Glomus fasciculatum Gigaspora margarita and Gigaspora gilmorie on plant growth and production of colchicine in Gloriosa superba and (2) determine if phosphate fertilizers would alter AM effects on colchicine production in Gloriosa superba.

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Material and Methods


Experimental Design
The Glass house study was carried out in year 2007, at the medicinal plant garden, B.H.U., Varanasi, India (25°18″N, 83°50″E). The experimental location experiences a semi-arid tropical climate. The soil of the experimental field was sandy loam texture, organic electrical conductivity 0.42 dS m−1, available carbon 0.38%, available nitrogen 180 kg ha−1, available phosphorus 21 kg ha−1 and pH 7.3. Plants used in the study were propagated from a V-shaped tuber of Gloriosa superba. One tuber was planted in each pot for rooting in the first week of May 2007. Tubers were sprouted in the month of July and healthy, profusely rooted, 30 days old plants were transplanted in the polybags of 1.5 litre volume each. After 180 days, December, 2007, plants were harvested from the polybags. Light irrigation was provided just after transplanting. The plants were allowed to grow and no fertilizer or pesticide was added to the soil during the course of the experiment. The aerial and underground tuber parts of the plants were separated and the different parts were washed with tap water, shade dried and kept in cellulose bags for further experiment. Two experiments were conducted: (1) Screening of best AM fungi influencing the growth and colchicine content of Gloriosa superba and (2) The second experiment were conducted to study effect of best AMF and different levels of P fertilizer [super phosphates (P2O5)] on growth and colchicine content of Gloriosa superba. The 2×4 Full factorial experiment was designed with two mycorrhizal conditions viz nonmycorrhizal and inoculated with Glomus mossae combined with four concentrations of phosphorus (0, 25, 50 and 100 mg kg−1 P-fertilizer) in the soil. The treatment combinations were arranged in a completely randomized design, with 10 plants per treatment. The experiment was done in triplicate.



Mycorrhizal Inoculation
Pot cultures of Glomus mossae, Glomus fasciculatum, Gigaspora margarita and Gigaspora gilmorei plants for 6 months. Five hundred grams of soil inoculum (50 spores/10 g soil), along with 200 mg of chopped AM Zea mays roots (AM colonization level 80%), was placed in furrows made with the help of a drill in each polybags before planting the rooted tuber of Gloriosa superba. Nonmycorrhizal inoculum consisted of 500 g rhizosphere soil and 200 mg chopped nonmycorrhizal sorghum roots obtained by sowing surface sterilized seeds of Zea mays in pot containing autoclaved soil.



Growth parameter and Nutrient estimation in Plants
Plants were grown under natural field conditions for 180 days. For each parameter studied, six plants were randomly harvested from polybags of each treatment. Roots were washed and dried on blotting paper. Fresh weight of shoot per plant was determined. Shoot samples were oven-dried at 72°C for 48 h for determination of dry weight (g/plant). Later, the same dried shoot samples were analyzed for their mineral concentration. Leaf sugar was extracted by the method of Angelov et al (1993). Total soluble sugar was determined using the method of Riazi et al (1985). Oven dried leaf was ground and sieved through 0.5 mm sieve. 0.2 g ground material was digested in a triple acid mixture (HNO3, H2SO4 and 60% HClO4 in a ratio of 10:1:4), for analysis of phosphorus. The amount of phosphorus in the digested sample was estimated by molybdenum blue method (Allen, 1989). The nitrogen content of leaf was measured using the semi-micro Kjeldahl method (Bremmer and Mulvaney, 1982). Leaf chlorophyll content was determined at harvest (n=3), by extraction of chlorophyll with acetone (Harborne, 1998). Procedure was modified as follows, representative semi-mature leaflets were collected and surface area was determined. Leaflets were placed in 5 mL of 80% acetone and stored in the dark for 7 d at 4 °C. Supernatant was quantified with a spectrophotometer (Perkins-Elmer UV/Vis Spectrophotometer), at 645 and 663 nm, and compared to an 80% acetone blank standard. Total chlorophyll content was expressed as mg/ g dry wt. of leaf.



Assessment of Arbuscular Mycorrhizal development
Assessment of roots for AM colonization was made at the end of the experiment by random sampling of roots. Percentage mycorrhizal root colonization was estimated following grid line intersect method (Giovanetti & Mossae, 1980) after staining the roots by the method of Philips & Hayman (1970).



Colchicine extraction and estimation
The amount of extracted colchicine from Gloriosa superba tuber was analyzed by high performance thin layer chromatography (HPTLC), as described by Bodoki et al, 2004 [26] with some modification. Standard colchicine and the samples were spotted on percolated silicagel F254 aluminum plate (E-Merck grade) as narrow bands 4 mm wide at a constant rate of 10 µl s−1 using Camag Linomat IV model applicator under nitrogen atmosphere. A mixture of toluene and methanol (85:15 v/v), was used as the mobile phase. For detection and quantification of colchicine (at Rf 0.2), scanning densitometry was performed using a Camag TLC scanner with CATS 4 software, in reflectance (at 360nm) and fluorescence modes (Hg lamp, 254 nm).



Statistical Design
The experiment was a 2 × 4 factorial in a completely randomized design with two AMF levels (AMF and Non-AMF), and four levels of P: 0, 25, 50, and 100 mg P Kg−1. There was one rooted Gloriosa superbaper polybags, with each polybag as a single replicate. Data were analyzed using General Linear Model by using MINITB (15 version) software. The numbers of replications were: growth data (n=9), P analysis (n=3), chlorophyll content (n=3), colchicines content (n=5, and AMF observations (n=675). Experiments were done in two run. All statistical analyses were performed with Statistical Package MINITAB (version 15).

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Results


Experiment 1: Screening of best AM fungi

 

Plant growth parameter, root colonization and nutrient concentration
Both the Glomus species successfully colonized the roots of Gloriosa superba (Table 1). No AM colonization was observed in those plant roots that were not inoculated with AM fungi. Percentage of root colonization varied between the fungi (Table I). AM fungal inoculation had a significant effect on all measured plant growth variables i.e tuber dry biomass, aerial shoot dry biomass and seed dry biomass (Table I). However, the level to which plant growth was enhanced varied between the fungal inoculants Glomus mossae-colonized plants performed consistently better than G. fasciculatum and other Am fungi-inoculated and non-mycorrhizal plants. Mycorrhizal plants consistently accumulated more quantities of nitrogen, sugar, chlorophyll and phosphorus in their leaf than the non-mycorrhizal plants. Glomus mossae inoculated plants performed better than other AM fungi inoculated plants. All AM fungi inoculated plants Glomus mossae, Glomus fasciculatum Gigospora margarita and Gigospora gilmorie resulted in root colonization while control plants did not show root colonization. However Glomus mossae found to be best among all the AM fungi inoculated plants (Table IA).


Table I-A

Screening of best AMF Inoculating Gloriosa superba and their effect on growth and nutrient.






AMF

Tuber
DM

Aerial
shoot
DM

Seed
DM

Leaf
Chl

Leaf
N(%DM)

Leaf
P.
(%DM)

Leaf Sugar
(%DM)

%Root
colonization





Control

37.33

15.6

0.51

3.11

1.39

0.21

2.11

0



G. mossae

51.6

23.5

1.22

4.21

1.65

0.31

2.87

92



G. fasciculatum

43.3

21.5

1.14

4.11

1.56

0.28

2.78

87



G. margarita

41.3

18.9

1.11

3.89

1.55

0.26

2.54

79



G. gilmorei

40.7

17.5

1.02

3.87

1.51

0.26

2.51

76







 

Concentration of colchicine in different parts of Gloriosa superba
Concentration of colchicine in the tuber, aerial shoot and seeds were significantly high in all AMF treatments compared to controls. The colchicine concentration was highest in Glomus mossae plants, which has significantly higher concentration of colchicine when compared with the other AMF inoculated plants. The next best treatments were Glomus fasciculatum, Gigospora margarita and Gigospora gilmorie treated Gloriosa superba plants (Table I-B).


Table I- B

Screening of best AMF Inoculating Gloriosa superba and their effect on colchicines content.






Amf Inoculation

Colchicine % dry
wt. Of shoot

Colchicine % dry
wt. Of root

Colchicine %
dry wt. Of
seed

Colchicine mg
Plant−1





Control

0.11

0.41

0.72

90



G. mossae

0.14

0.53

0.94

190.34



G. fasciculatum

0.13

0.48

0.9

150.4



G. margarita

0.12

0.45

0.88

125.67



G. gilmorei

0.11

0.43

0.86

121.21









Effect of Glomus mossae and different level of phosphorus fertilizers on Gloriosa superba

 

Plant Growth, root colonization and nutrient concentration in Gloriosa superba
AM fungal inoculation and/or phosphorus fertilization had a significant effect on all measured plant growth variables (Table II A). However, the level to which plant growth was enhanced varied between the different levels of P applications. Glomus mossae-colonized plants performed consistently better than non-mycorrhizal P-fertilized plants. Mycorrhizal inoculation of plants in the 25 mg kg−1P-soil further increased the tuber and aerial dry biomass and found to be best when compared with the non-mycorrizal plants treated with different levels of P fertilizers and mycorrhizal plants treated with different levels of P fertilizers. The treatment GM + 25 mg kg−1P produced up to 51% more tuber biomass than non-inoculated control plants (Fig II-A and II-B). Mycorrhizal plants with different level of P consistently accumulated more quantities of phosphorus, nitrogen and sugar in their leaves than the non-mycorrhizal plants with different level of P. However, the differences were not significant in plants grown in P-fertilized soil (Table II A and  andB).B

 

). Glomus mossae + 25 mg P applied plants resulted in higher concentrations of P, N and sugar in leaves when compared with the other AMF+ P and P applied plants. Mycorrhizal inoculation + P-fertilizer and P-fertilizer application alone significantly influence the concentrations of chlorophyll. Among AM fungi + P-fertilizer together AMF + 25 mg P resulted in significant increases in concentrations of chlorophyll compared to non AMF + P fertilizer applied plants and AMF + P applied plants. All AM fungi inoculated plants with Glomus mossae + P applied plants resulted in root colonization while non -AMF + P applied plants did not show root colonization. However Glomus mossae+ 0 mg P applied plants showed maximum root colonization and found to be best among AMF + P applied plants. (Fig IIA and  andBB)
 

 

Figure II-A

Main effect plot of different AM fungi and phosphorus on foliar nutrient parameter of Gloriosa superba

 

FigureII-B

Graph of different AM fungi and phosphorus on foliar nutrient parameter of Gloriosa superba





Table II- A

Effect of phosphorus and arbuscular mycorrhizal fungi (AMF) on growth and nutrient of Gloriosa superba plants.






TCP

AMF

Tuber
DM

Aerial
shoot
DM

seed

Leaf
N(%DM)

Leaf P.
(%DM)

Leaf Sugar
(%DM)

Chlorophyll

Root
colonization





0

No

36.16

15.5

0.54

1.22

0.18

2.11

3.11

0



25

No

39.66

17.3

0.62

1.33

0.24

2.33

3.24

0



50

No

41.66

17.9

0.72

1.41

0.27

2.43

3.23

0



100

No

41.23

17.8

0.64

1.36

0.25

2.32

3.11

0



 

 

 

 

 

 

 

 

 

 



0

Yes

52.00

23.5

0.54

1.65

0.31

2.87

4.23

92



25

Yes

54.66

24.3

1.34

1.89

0.39

2.97

4.17

88



50

Yes

58.60

27.6

1.21

1.79

0.35

2.93

4.16

86



100

Yes

56.50

25.7

1.12

1.77

0.34

2.91

4.16

77



P

AMF

***

**

 

**

**

*

**

**



 

P

**

***

 

**

**

**

**

*



 

AMFX P

*

*

 

**

*

*

*

**





Significance according to ANOVA, NS, *, **, ***, non-significant and significant P. 0.05, 0.01, 0.001, respectively Means (n=3).




Table II- B

Effect of phosphorus and arbuscular mycorrhizal fungi (AMF) on colchicines content of Gloriosa superba plants






Phosphorus

G. mossae

Colchicine %
dry wt. of
shoot

Colchicine % dry wt.
of tuber (root)

Colchicine %
dry wt. of seed

Colchicines mg
plant−1





0

No

0.09

0.44

0.78

177.87



25

No

0.12

0.46

0.81

179.97



50

No

0.14

0.51

0.81

184.7



100

No

0.14

0.49

0.76

178.9



 

 

 

 

 

 



0

Yes

0.12

0.56

0.94

342.6



25

Yes

0.14

0.65

0.96

348.9



50

Yes

0.16

0.61

0.93

345.7



100

Yes

0.15

0.61

0.91

343.8



P

AMF

**

*

***

**



 

P

**

**

**

**



 

AMFXP

**

*

*

**





Significance according to ANOVA, NS, *, **, ***, non-significant and significant P. 0.05, 0.01, 0.001, respectively. Means (n=3).




 

Concentration of cochicine in different parts of Gloriosa superba plants
Concentration of colchicine in the tuber was significantly higher in all AMF+P treatments compared to controls and non AMF+P treated plants. The colchicine concentration was highest in Glomus mossae+25 mg P treated plants, which has significantly higher concentration of colchicine when compared with the different levels of P+AMF inoculated plants and different levels of P+ non AMF plants. The total colchicine content of plant (mg / plant), was significantly high in plants inoculated with Glomus mossae and 25 mg kg−1phosphorus fertilizer (348.9 mg /plant), while the control contain least colchicine (177.87 mg / plant), (Fig.IIIA and  andB).B). The study suggests a potential role of AM fungi in improving the concentration of colchicine in Gloriosa superba tuber.

 

Figure III-A

Main effect plot of different AM fungi and phosphorus on colchicine content of Gloriosa superba

 

 

Figure III-B

Graph of different AM fungi and phosphorus on colchicine content of Gloriosa superba

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Discussion
The results clearly demonstrate high AM effectiveness in increasing the plant growth. The response of G. mossae was better than G. fasciculatum and other AMF inoculated plants. The effect of mycorrhiza on plant development is influenced mainly by both the host plant and fungal partner (Bethlenfalvay et al., 1989). Jackobsen et al. (1992) showed that different isolates of AM fungi can result in different effects on plant growth. The P-concentration of mycorrhizal plant varied with AM fungal inoculum. The results indicate marked differences in capacity of fungal species for the uptake of P by hyphae. This finding is in accordance with observations of Pearson and Jakobsen (1993). The colchicine content also varies among plants inoculated with different strains of AM Fungi. This finding is in accordance with Abu-Zeyad et al (2005) observed that castanospermine content in Castanospermum australe root are enhanced in the presence of AMF. Pandey and Banik (2009) found Glomus mossae to be the efficient AM fungi involved in enhancing growth and barbaloin content in Aloe vera
A significant increase in growth and colchicine concentration in Gloriosa superba was recorded in plants from all treatments relative to the controls. Increased growth and development in AM plants compared to non-mycorrhizal ones has been reported for many plant species, including members of Liliaceae (Bryla and Duniway 1997; Ultra et al. 2007). Different effects on plant development were observed depending on the fungal species and P-status of the soil. Glomus mossae-inoculated plants registered higher yield at different levels of phosphorus in soil. The greatest growth effect was observed with Glomus mossae in the P-enriched (50 mg kg−1) soil. The plant given combined treatment of phosphate and mycorrhizal inoculation could utilize the maximum benefits of phosphate fertilizer inspite of their decreased AM colonization in roots. Mycorrhization or P-fertilization alone did not influence the concentration of photosynthetic pigments. These results are not in agreement with results obtained in some earlier works (Giri et al. 2003; Kapoor and Bhatnagar 2007). Taiz and Zeiger (1998) correlated enhanced concentrations of chlorophyll to Cu uptake. Cu is involved in the electron transport system and is a component of the chlorophyll protein plastocyanin. Thus, significant differences in concentrations of chlorophyll-a and chlorophyll-b observed in the present study may be attributed to similar Cu concentrations in leaves of AM-inoculated and nonmycorrhizal plants.
Colchicine has been reported to accumulate in tuber and other parts of the Gloriosa superba plants (Ferreira and Janick 1995). Therefore, substances that improve tuber and shoot growth are presumed to increase colchicine yield. Moreover, biosynthesis of colchicine is dependent on primary metabolism, e.g., photosynthesis and oxidative pathways for carbon and energy supply (Singh et al. 1990). According to Fitter (1988) and Giri et al. (2003), net photosynthesis of mycorrhizal plants can increase as a result of improved plant nutritional status. Factors that increase dry matter production may influence the interrelationship between primary and secondary metabolism, leading to increased biosynthesis of secondary products (Shukla et al. 1992). It appears that significant improvement in plant biomass results in greater availability of substrate for colchicine biosynthesis (van Gelgre et al. 1997). The enhanced concentration of colchicine by mycorrhization and/or P-fertilization may be due to improved growth and nutrient status of the plants. Consequently, the increase in colchicine concentration depends on the species of AM fungus used, with G. mossae being a more efficient symbiont for Gloriosa superba than G. fasciculatum and other species of Gigaspora.
In conclusion, the present study demonstrates the effectiveness of AM fungi in improving the concentration of colchicine in Gloriosa superba. Inoculation with suitable AM fungal species along with P-fertilization produces significant increases in tuber biomass production, resulting in significant increases in colchicine content of individual plants. Thus, introduction of mycorrhizal technology in conjunction with P-fertilizers will be helpful in developing low-cost cultivation of Gloriosa superba and production of colchicine.


​
 

 

Figure I-A

Main effect plot of different AM fungi and phosphorus on growth parameters and mycorrhization of Gloriosa superba

 

Figure I-B

Graph of different AM fungi and phosphorus on growth parameters and mycorrhization of Gloriosa superba

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Acknowledgements

This research was funded by the Council of Scientific and Industrial Research, New Delhi, India.


 

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References

1. Allen SE. Chemical analysis of ecological materials. 2nd edn. Oxford, London: Blackwell Scientific Publishers; 1989.
2. Angelov MN, Sun J, Byrd GT, Brown RH, Black CC. Novel characteristics of cassava, Manihot esculenta Crantz, a reputed C3-C4 intermediate photosynthetic species. Photosynth Res. 1993;38:61–72.[PubMed]
3. Alexander P, Brigitte N, Meinhart Z. Immuno assays for the quantitative determination of colchicines. Planta Medica. 1994;60:77–83. [PubMed]
4. Bellet P, Gaignault, Le JC. Gloriosa superba L. et al production de substances colchiciniques. Ann Pharm Fr. 1985;43:345–347. 1985. [PubMed]
5. Bodoki E, Oprean R, Vlase L, Tamas M, Sandulescu R. Fast determination of colchicine by TLC-densitometry from pharmaceuticals and vegetal extracts. J of Pharmand Biomed Anal. 2005;37:971–977.[PubMed]
6. Bremmer JM, Mulvaney CS. Nitrogen-total. In: Page AL, Miller RH, Keeney DR, editors. Methods of soil analysis: chemical and microbiological properties Part 2. 2nd edn. Madison: American Society of Agronomy; 1982. pp. 595–624.
7. Bryla DR, Duniway JM. Effects of mycorrhizal infection on drought tolerance and recovery in safflower and wheat. Plant Soil. 1997;197:95–103.
8. Clewer HWV, Green SS, Tutin F. The constituents of Gloriosa superba. J Chem Soc. 1915;107:835–846.
9. Ferreira JFS, Janick J. Floral morphology of Artemisia annua with special reference to trichomes. Int J Plant Sci. 1995;156:807–815.
10. Finnie JF, van Staden J. In vitro production of Sandersonia and Gloriosa. Plant Cell Tiss Organ Cult. 1989;19:151–158.
11. Finnie JF, van Staden J. Isolation of colchicine from Sandersonia aurantica and Gloriosa superba. Variation in the alkaloid levels of plants grown in vivo. J Plant Physiol. 1991;138:691–695.
12. Finnie JF, Van Staden J. Gloriosa superba L. (Flame Lily): micropropagation and in vitro production of colchicines. In: Bajaj YPS, editor. Biotechnology in Agriculture and Forestry, vol 26: Medicinal and Aromatic Plants VI, (Chapter X) 1994. pp. 146–166.
13. Fitter AH. Water relations of red clover Trifolium pretense L. as affected by VA mycorrhizal infection and phosphorus supply before and during drought. J Exp Bot. 1988;202:595–603.
14. Shukla A, Abad Farooqi AH, Shukla YN, Sharma S. Effect of triacontanol and chlormequat on growth, plant hormones and artemisinin yield in Artemisia annua L. Plant Growth Regul. 1992;11:165–171.
15. Giovannetti M, Mosse B. An evaluation of techniques for measuring vesicular and arbuscular mycorrhizal infection in roots. New Phytologist. 1980;84:489–500.
16. Giri B, Kapoor R, Mukerji KG. Influence of arbuscular mycorrhizal fungi and salinity on growth, biomass and mineral nutrition of Acacia auriculiformis. Biol Fertil Soils. 2003;38:170–175.
17. Goldfinger SE. Colchicine for familial Mediterranean fever. J Med. 1972;287:1302. [PubMed]
18. Harborne JB. Recommended techniques, chlorophyll estimation. In: Harborne JB, editor. Phytochemical methods. London, UK: 1973. pp. 205–207.
19. Hayashi T, Yoshida K, Sano K. Formation of alkaloids in suspension cultured Colchicum autumnale. Phytochemistry. 1988;27:371–1374.
20. Kapoor R, Bhatnagar AK. Attenuation of cadmium toxicity in mycorrhizal Celery (Apium graveolensL.) World J Microbiol Biotechnol. 2007;23:1083–1089.
21. Kapoor R, Giri B, Mukerji KG. Glomus macrocarpum: a potential bioinoculant to improve essential oil quality and concentration in dill (Anethum graveolens L.) and carum (Trachyspermum ammi (Linn.) Sprague) World J Microbiol Biotechnol. 2002a;18:459–463.
22. Kapoor R, Giri B, Mukerji KG. Mycorrhization of coriander (Coriandrum sativum L.) to enhance the concentration and quality of essential oil. J Sci Food Agric. 2002b;88:1–4.
23. Kapoor R, Giri B, Mukerji KG. Improved growth and essential oil yield and quality in Foeniculum vulgare Mill. on mycorrhizal inoculation supplemented with P-fertilizer. Bioresour Technol. 2004;93(3):307–3011. [PubMed]
24. Kershenobich D, Varga F, Garcia Tao G, Tamayo RP, Gent M, Rojkind M. Colchicine in the treatment of cirrhosis of the liver. N Engl J Med. 1988;318:1709–1713. [PubMed]
25. Khaosaad T, Vierheilig H, Nell M, Zitterl-Eglseer K, Noval J. Arbuscular mycorrhiza alter the concentration of essential oils in oregano (Origanum sp., Lamiaceae) Mycorrhiza. 2006;16:443–446.[PubMed]
26. Owusu-bennoah E, Mossae B. Plant growth responses to vesicular-arbuscular mycorrhiza. XI. Field inoculation responsesin barley, Lucerne and onion. New phytologist. 1979;83:133–140.
27. Pandey DK, Banik RM. The Influence of Dual Inoculation with Glomus mossae and Azotobacter on Growth and Barbaloin Content of Aloe vera. Am-Eu J Sust Agric. 2009;3(4):703–714.
28. Phillips JM, Hayman DS. Improved procedures for clearing roots and staining parasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection. Trans Br Mycol Soc. 1970;55:158–160.
29. Riazi A, Matsuda K, Aslan A. Water stress induced changes in concentration of praline and other solutes in growing regions of young barley leaves. J Exp Botany. 1985;172:1716–1725.
30. Roberts WN, Liang MH, Stern SH. Colchicine in acute gout: reassessment of risks and benefits. J Am Med Assoc. 1920;257:2. [PubMed]
31. Sailo GL, Bagyaraj DJ. Influence of different AM-fungi on the growth, nutrition and forskolin content of Coleus forskohlii. Mycol Res. 2005;109:795–798. [PubMed]
32. Sarin YK, Jamwal PS, Gupta BK, Atal CK. Colchicine from seeds of Gloriosa superba. Curr Sci. 1977;43:87.
33. Singh N, Luthra R, Sangwan RS. Oxidative pathway of essential oil biosynthesis in the developing Cymbopogon flexuosus leaf. Plant Physiol Biochem. 1990;28:703–710.
34. Smith FA, Jakobsen I, Smith SE. Spatial differences in acquisition of soil phosphate between two arbuscular mycorrhizal fungi in symbiosis with Medicago truncatula. New Phytol. 2000;147:357–366.
35. Srivastava UC, Chandra V. Gloriosa superba Linn. (kalihari) an important colchicine producing plant. J Res Ind Med. 1977;10:92–95.
36. Taiz L, Zeiger E. Plant physiology. 2nd edn. Sunderland: Sinauer; 1998. pp. 251–286.
37. Trease SE, Evans D. Pharmacognosy, Colchicum Seed and Corm. 12th Edn. London, UK: WB Saunders Co. Ltd; 1983. pp. 593–597.
38. Ultra V, Tanaka S, Sakurai K, Iwasaki K. Effects of arbuscular mycorrhizal and phosphorus application on arsenic toxicity in sunflower (Helianthus annuus l.) and on the transformation of arsenic in the rhizosphere. Plant Soil. 2007;290:29–41.
39. Van Gelgre E, Vergauwe A, van den Eeckhout E. State of the art of the production of the antimalarial compound artemisinin in plants. Plant Mol Biol. 1997;33:199–209. [PubMed]
40. Yoshida K, Hayashi T, Sano K. Colchicoside in Colchicum autumnale bulbs. Agric Biol Chem. 1988a;52:593–594.
41. Yoshida K, Hayashi T, Sano K. Colchicine precursors and the formation of alkaloids in suspension cultured Colchicum autumnale. Phytochem. 1988b;27:1375–1378.


 

Articles from African Journal of Traditional, Complementary, and Alternative Medicines are provided here courtesy of African Traditional Herbal Medicine Supporters Initiative

[peacefrog]

DAMN nice thread Mikey. Sorry. I guess I need to look around this place more.

Great information and nicely done. Bow, my friend.

[coorsmikey]

This thread is chopped up and needs to be re-written. There is a lot of drama and bantering that is hidden because some members felt it was unsafe to for me to talk about colchicine and extraction of colchicum. Some pretty heated arguments from mainly the fear of the unknown that actually caused me to lose interest in sharing the research here at all or even to continue documenting it. Some things are sometimes better left unsaid, I suppose.

Edited by coorsmikey, 20 February 2019 - 05:00 PM.

[peacefrog]

Understood. Well, still an awesome read my friend.

[PistolPete13]

I had completely missed this, saw it for the first time last week. If you get some time to get around to that re-write, I will be eagerly reading.

 

Thanks!

[PistolPete13]

My initial thoughts after having a read then a quick look into the literature......I would not completely rule out the possibility that you may well have induced polyploidy in that old APE and created a neopolyploid(synthetic polyploid). If one were to just collect and germinate spores with no real pattern I would imagine you would get a natural stabilization(not degradation) of the strain. Since PE is a mutant(and also horrible spore producer) you would see a disappearance over time and reverting back to the more stable spore producing PF albino phenotypes, unless you stepped in and started selecting for those traits. I saw comments like;

 

 

This reminded me of early generations of F Albino created by TheChosenOne.

 

And I know polyploidy in fungi is not unheard of, and in all things usually results in things like faster more aggressive growth, higher potency ect.

 

But then I am reading about the phenotypes and scratching my head.

 

Cover me, Im going in.............to the library I have some reading to do.....[about the effects of colchicine on the fungal cell wall, and if somatic hybridization of fungi has been accomplished with anything similar].

[coorsmikey]

My initial thoughts after having a read then a quick look into the literature......I would not completely rule out the possibility that you may well have induced polyploidy in that old APE and created a neopolyploid(synthetic polyploid). If one were to just collect and germinate spores with no real pattern I would imagine you would get a natural stabilization(not degradation) of the strain. Since PE is a mutant(and also horrible spore producer) you would see a disappearance over time and reverting back to the more stable spore producing PF albino phenotypes, unless you stepped in and started selecting for those traits. I saw comments like;

 

 

This reminded me of early generations of F Albino created by TheChosenOne.

 

And I know polyploidy in fungi is not unheard of, and in all things usually results in things like faster more aggressive growth, higher potency ect.

 

But then I am reading about the phenotypes and scratching my head.

 

Cover me, Im going in.............to the library I have some reading to do.....[about the effects of colchicine on the fungal cell wall, and if somatic hybridization of fungi has been accomplished with anything similar].

Please add you findings PistolPete! You do have knack for finding some good papers.

 

I would agree with the neoplolyploid APE, but all resulting offspring did come directly from spores of normal pigmented fruits.

Edited by coorsmikey, 16 February 2019 - 10:29 PM.

[PistolPete13]

It is going to be interesting, whatever it was! And I also vaguely recall something about colchicine messing some part of the development of the cell wall in fungi, which would be relevant considering they were trying to grow across it. Anyway, I will try and see if anything more solid comes up......

Edited by PistolPete13, 16 February 2019 - 10:43 PM.

[CatsAndBats]

Damn, y'all been busy...   ;)

[PistolPete13]

So I have had a look and I cannot find anything that would explain this other than some extremely random occurrence that would probably not be reproducible.

There is quite a bit of research on the effects of colchicine on fungi, but they are for the most part focused on chromasome doubling.

 

And you can see from this paper on oyster mushrooms it seems to be the goto method for trying to induce polyploidy in fungi;

 

Nuclear abnormality in the mycelia of Pleurotus ostreatus in presence of colchicine                                                                                                                                                                                                                                                                                     At first, we investigated the conditions of the
coichicine treatment in order to produce the au-
topolypoids in the fungus.                                                                                                                                                                       toyama1994.pdf   635.36KB   360 downloads

Something that did pop up that I believe belongs in this thread which is 'haploid doubling', just quickly when cells divide they do so by making copies of themselves. They make identical copies of their chromosomes which are attached to each other and during mitosis the chromosomes are separated into two nuclei;

Cochicine inhibits mitosis and the cell is unable to separate the two chromosomes as it would normally do, and is stuck(literally) with twice the number it should have. That is an extremely basic rundown of how colchicine works;

Spores germinate haploid(one chromosome, half of what is needed) it needs to find another compatible haploid that it can share genetic information with(and gain a second chromosome) and become a dikaryon(which is an intermediary phase, where two haploid chromosomes cohabit before fusing). When they are ready they fuse to form a diploid, which can fruit and complete its life cycle and pass on its DNA.

Colchicine treatment of haploids is referred to as chromosome doubling or haploid doubling, where for example a single haploid spore is germinated and a culture taken from it and grown on colchine. Which doubles its chromosome producing a haploid individual theoretically capable of fruiting.

Cubensis has what are called mating types, which is basically a safegaurd against serious inbreeding.

"Only individuals with different specificities are compatible with each other and therefore able to start the mating event."

So if the alleles match up they are not compatible, and hence it avoids for the most part a lot of genetic dead ends(which is bad for long term survival) that comes along with inbreeding. It mostly avoids for example matching up recessive traits which would allow full expression of them, which could lead to genetic degradation. Or if the recessive trait is poor sporulation this is not a good thing for survival, therefore masking or suppressing these potentially dangerous things makes them able to produce much more genetically stable offspring by mating with themselves(which would not go as well for humans for example who dont have these defenses).

Haploid doubling to produce a diploid from a spore, cheats every rule of the mating type that is coded into their DNA. And would produce some funky individuals! In a normal multi spore you see aborts, blobs, individuals unable to fruit, so taking away their safety net would make things worse. But you would eventually produce some very desirable individuals, that would never be seen naturally(apart from those 1 in a million freaks).

They use this in plant breeding for a reason!


 

[Microbe]

So I have had a look and I cannot find anything that would explain this other than some extremely random occurrence that would probably not be reproducible.
There is quite a bit of research on the effects of colchicine on fungi, but they are for the most part focused on chromasome doubling.

And you can see from this paper on oyster mushrooms it seems to be the goto method for trying to induce polyploidy in fungi;

Nuclear abnormality in the mycelia of Pleurotus ostreatus in presence of colchicine At first, we investigated the conditions of the
coichicine treatment in order to produce the au-
topolypoids in the fungus. toyama1994.pdf

Something that did pop up that I believe belongs in this thread which is 'haploid doubling', just quickly when cells divide they do so by making copies of themselves. They make identical copies of their chromosomes which are attached to each other and during mitosis the chromosomes are separated into two nuclei;


Cochicine inhibits mitosis and the cell is unable to separate the two chromosomes as it would normally do, and is stuck(literally) with twice the number it should have. That is an extremely basic rundown of how colchicine works;

Spores germinate haploid(one chromosome, half of what is needed) it needs to find another compatible haploid that it can share genetic information with(and gain a second chromosome) and become a dikaryon(which is an intermediary phase, where two haploid chromosomes cohabit before fusing). When they are ready they fuse to form a diploid, which can fruit and complete its life cycle and pass on its DNA.

Colchicine treatment of haploids is referred to as chromosome doubling or haploid doubling, where for example a single haploid spore is germinated and a culture taken from it and grown on colchine. Which doubles its chromosome producing a haploid individual theoretically capable of fruiting.

Cubensis has what are called mating types, which is basically a safegaurd against serious inbreeding.

"Only individuals with different specificities are compatible with each other and therefore able to start the mating event."

So if the alleles match up they are not compatible, and hence it avoids for the most part a lot of genetic dead ends(which is bad for long term survival) that comes along with inbreeding. It mostly avoids for example matching up recessive traits which would allow full expression of them, which could lead to genetic degradation. Or if the recessive trait is poor sporulation this is not a good thing for survival, therefore masking or suppressing these potentially dangerous things makes them able to produce much more genetically stable offspring by mating with themselves(which would not go as well for humans for example who dont have these defenses).

Haploid doubling to produce a diploid from a spore, cheats every rule of the mating type that is coded into their DNA. And would produce some funky individuals! In a normal multi spore you see aborts, blobs, individuals unable to fruit, so taking away their safety net would make things worse. But you would eventually produce some very desirable individuals, that would never be seen naturally(apart from those 1 in a million freaks).

They use this in plant breeding for a reason!

Creating a double haploid or doubling the chromosomes in a single monokaryotic culture is something i was researching but i needed a break. Needles was supposed to be grabbing a single spore and germinating for me as he has the tools to do it alot easier then i can. I would have to rely on a dilution method and 100's of agar plates which im just not prepared to commit to.

If successful it would open the doors wide open for selective breeding including crossing or creating hybrids by being able to truly control the lineage.



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[PistolPete13]

 

Creating a double haploid or doubling the chromosomes in a single monokaryotic culture is something i was researching but i needed a break.

 

That is very interesting, I would love to hear where your research led.

 

 

If successful it would open the doors wide open for selective breeding including crossing or creating hybrids by being able to truly control the lineage.

 

Exactly, overcoming the mating types and achieving complete homozygosity would be a game changer!

 

I have noticed that a dilution onto agar followed by lowering the incubation temp, makes the germination play out a lot slower and you would have a bigger window to grab cultures as they germinate.