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OfflineMahoney
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Induce polyploidy
    #196213 - 02/15/09 01:37 PM (5 years, 2 months ago)

I found this interesting and thought I'd share it with you.

Introduction:
Colchicine is a toxic chemical that is often used to induce polyploidy in plants. Basically, the colchicine prevents the microtubule formation during cell division, thus the chromosomes do not pull apart like they normally do. The end result is a cell that now has double the number of chromosomes that it would normally have. If this cell divides again in the future, then the doubled number of chromosomes are passed to the offspring cells. Plants that have more than the normal two sets of chromosomes are termed “polyploidy” in general, although specific names are given to the certain chromosome numbers (e.g. tetraploid or 4N plants have four sets of chromosomes).
Polyploid plants are generated in an effort to create new plants that have new characteristics. Sometimes the polyploidy plants are sickly and not viable, but sometimes the polyploid plants have larger leaves and flowers. Orchid growers will often sell polyploidy plants that are larger or have larger flowers. Often the polyploid nature of the plant is included in the cultivar name (G. species ‘big flower 4N’). Colchicine is also used to try to make fertile hybrids between species with different numbers of chromosomes. The use of colchicine to make hybrids is well documented in ICPS cultural section of this web site.
Unfortunately, colchicine is very toxic and hazardous to handle. It is acutely toxic and has been responsible for many accidental poisonings by people or pets consuming the “autumn crocus” (Colchicum sp.) that are sometimes used in gardens. In addition to its acute toxicity, it also causes chromosomal defects. Overall, this chemical in it pure form is best handled in a chemistry lab with a fume hood with gloves and other personal protective equipment. Once it is dissolved and diluted into water, it can be handled outside the fume hoods but gloves are still a necessity since the chemical may be adsorbed through the skin. A more comprehensive review of colchicine toxicity can be found in the cultural of this web site .
The objective of this project was to assess what concentrations of colchicine are necessary to have biological effects on different carnivorous plant species. Seeds of different CP species were obtained from the seed bank and divided into different treatments. For all species, there was a “control group” and a 500 ppm colchicine treatment group. Some species with abundant seed availability had additional treatment concentrations. The number of germinations were then counted after it was clear the control group no longer had any new germinations, which was two months for most species. The goal is to create tetraploid plants with different characteristics, but the confirmation of polyploidy requires chromosome counting, which is beyond the scope of this project.
Methods:
Seeds from 5 different genera were obtained from the ICPS seed bank. The seed from each species was homogenized to prevent inter-packet variation. The seed was counted into equal number of seeds for each treatment. For each treatment, the seed was divided into three equal portions which were treated separately. The one exception was the Dionaea seed where each treatment was dosed together.
The dosing procedure involved creating a concentrated colchicine solution by adding 0.51 g of colchicine into 100 mL of purified (HPLC-grade) water. This created a “stock” solution of 5100 ppm colchicine by weight. For each treatment, the stock solution was diluted to achieve the desired concentration of colchicine. For example, the 510 ppm solution involved diluting 1 mL of the stock solution by adding 9 mL of purified water in a test tube. The control group just had 10 mL of pure water added to it. The seeds were added to the test tubes and they were allowed to soak in the solution for 96 hour. The test tubes were wrapped in aluminum foil to protect the solutions from light since colchicine breaks down in light. They were stored at room temperature for the 96 hours. The solutions were shaken each day to make sure that the seeds were in contact with the solution since many of the seeds initially floated on the solution. The Sarracenia seed were stratified in a refrigerator for 4 weeks before being dosed with colchicine.
The seeds were planted into 4” (~10 cm) square pots that were filled with a mixture of 50 sand and 50% peat (“CP mix”) except for the Darlingtonia that were planted on pure sphagnum. The pots were individually placed in plastic bags and then the solution with seeds in it was poured into the pots as to avoid coming in contact with the toxic solution. The test tubes were rinsed with clean water and poured into the pots to wash out any remaining seeds into the pots. The plastic bags were sealed and the pots were placed in a greenhouse or sunny windowsill for germination.
The seed germination was monitored weekly. Once the number of new germinations in the control group ceased, then a final count of the number of seedlings was conducted. The germination counts did include plants that were probably not viable in the long run. These stunted seedlings were noted. For example, Figure 1 shows the normal and stunted seedlings for Byblis liniflora. Any seedling that died before the final count was not counted as a “germination”. The pots were counted twice and the two counts were averaged to get the final number of seedlings. The Darlingtonia pots were the exception in that they were scored the following spring about 6 months after germination. The Darlingtonia score does not include plants that died within the 6 month period.


Results:
The results from the colchicine treatment indicates that there is a high degree of inter-genera susceptibility to colchicine as applied in this dosing experiment (Table 1). None of the Drosera species showed any visible effect from the colchicine; the germination was comparable to the control group and the seedlings looked normal. These plants, due to their short life cycle, were monitored until some of the plants started blooming. Almost none of the plants looked different from “normal” plants of the same species except that a few of the D. capensis looked a little more washed-out in terms of color, but this is qualitative at best. Two of the D. binata plants looked larger than the control, so they were isolated and they will be evaluated for differences in growth rate. If these plants seem to be larger in the long run, then chromosome counts will be conducted on them to verify if polyploidy were generated.
On the other extreme, Byblis liniflora showed a very high degree of susceptibility to colchicine. The germination and survival rate among seedlings was very low in the treated group. Furthermore, the seedlings were clearly stunted and most were not viable (Figure 1). Some of the colchicine seedlings developed a few stunted leaves but then died. These plants were probably polyploid, but the lack of plant viability prevented testing for chromosome counts. Any future attempts at making polyploid Byblis liniflora will need to use lower concentrations of colchicine.
The Sarracenia leucophylla showed no observable effect from the colchicine treatment. The treated group had effectively the same germination rate as the controls and the seedlings appeared to be normal. Future experiments to induce polyploidy will need to use higher concentrations of colchicine.
The Darlingtonia seedlings germinated and where then scored in the following spring approximately 6 months after germination, thus these scores represent both germination as early survival. The data suggests that colchicine may have reduced the germination/seedling viability, but the rather small sample set (57 control seeds and 57 treated seeds) makes any comparison rather tenuous.
Lastly, the Dionaea showed an effect from the colchicine treatment, but not as dramatic as the Byblis. The germination rate of both of the treated groups was lower than the control group by almost a factor of two. The higher colchicine treatment also started to generate clearly stunted plantlets that were comparable to the Byblis plantlets. It would appear that 5100 ppm colchicine would be a good dosing concentration for future experiments since germination was still reasonable (albeit ½ of normal) and some of the plants were clearly affected. Whether any of these plants will develop into anything of cultural interest remains to be seen. If any of the plants are visibly different, then they will be tested for polyploidy.
The ultimate objective of this experiment is to generate polyploid plants with desirable characteristics. Since chromosome counts require considerable effort, only plants that appear to be different will be tested in the future. The long development times for some species (e.g. Sarracenia, Dionaea) means that some species may take considerable time to show effects. Although some plants in this test were abnormal (Byblis, Dionaea, Drosera), none of them have been confirmed as polypoid and abnormal Byblis were not viable. Until some of the plants are confirmed as polyploid, the results of this experiment are best used as a “range-finding” experiment that determines what concentrations of colchicine are needed to produce effects without causing excessive seedling mortality. Byblis needed dosing concentrations below 500 ppm while Darlingtonia and Dionaea produce biological effects at 500 ppm dosing concentration. Drosera and Sarracinea require dosing concentrations of greater than 1000 ppm or a longer dosing exposure time.
Additional experiments may be conducted in the future to add more species to the colchicine susceptibility table and to generate new plants with different characteristics (hopefully positive!). If such experiments are conducted in the same fashion again, they will be added to the table presented here.

Table 1. Germination success (%) for several species of carnivorous plants exposed to different concentrations of colchicine (in ppm) for 96 hours. The number of seeds per each treatment group is given after the species name.
Species (number of seeds in each treatment) Germination success (%) by
colchicine concentration in ppm

    * 0 (control) 102 510 2550 5100
    * ________________________________________ ________________________________________
    * Byblis liniflora (n = 150) 20.6 --- 1.3 a --- ---
    *
    * Darlingtonia californica (n = 57) 33.3 (B ) 17.5 b
    *
    * Dionaea muscipula (n = 96) 38.5 --- 22.3 --- 18.2 (a)
    *
    * Drosera binata (n = 150) 35.3 34.7 --- ---
    * Drosera capensis ‘red’ (n = 300) 67.5 74.0 69.7 65.0 ---
    * Drosera filiformis “Florida red” (n = 150) 54.0 --- 50.0 ---
    *
    * Sarracinea leucophylla (n = 150) 48.7 --- 50 --- ---



(a) some germinating seedlings were clearly stunted and were not viable.
(b ) The seedling success was scored in the following spring approximately 6 months after germination.

Thomas Cahill
Arizona State University


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Offlinet0ad
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Re: Induce polyploidy [Re: Mahoney]
    #196226 - 02/15/09 02:44 PM (5 years, 2 months ago)

ow... my head hurts now... :frown:


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Offlineneopet nub

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Re: Induce polyploidy [Re: Mahoney]
    #196231 - 02/15/09 02:55 PM (5 years, 2 months ago)

Polyploidy is bad for the average grower.  It kill's off a large amount of seeds and very few people even give the plant the correct settings to show a substantially increased yield that would make polyploidy a useful process.


PP on Canni

"...attempted inducing polyploidy in many varieties, none leading to agronomic success."

Also, shocking and stressing your plants using this process can kill them off, if not slowing the growing process down tremendously.


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OfflineMahoney
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Re: Induce polyploidy [Re: neopet nub]
    #196560 - 02/16/09 11:43 AM (5 years, 2 months ago)

Yeah but it's fun.


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Offlinesnufkin
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Re: Induce polyploidy [Re: neopet nub]
    #259073 - 08/03/09 08:45 AM (4 years, 8 months ago)

Quote:

neopet nub said:
PP on Canni

"...attempted inducing polyploidy in many varieties, none leading to agronomic success."





http://en.wikipedia.org/wiki/Polyploid#Polyploidy_in_plants
Quote:

Polyploid plants tend to be larger and better at flourishing in early succession habitats such as farm fields.




I have never attempted to induce polyploidity with chemicals but in both of my grows one (usually a sativa) turned out polyploid and had an unexpectedly high yield.

Here's an example of a triploid:


--------------------
Grows: 1 2


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Re: Induce polyploidy [Re: snufkin]
    #259231 - 08/03/09 08:30 PM (4 years, 8 months ago)

Polyploidy is a neat word.  It sounds like one a child would make up.  Interesting read.  Thanks for the bump!


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Re: Induce polyploidy [Re: snufkin]
    #259250 - 08/03/09 08:50 PM (4 years, 8 months ago)

Quote:

snufkin said:
Quote:

neopet nub said:
PP on Canni

"...attempted inducing polyploidy in many varieties, none leading to agronomic success."





http://en.wikipedia.org/wiki/Polyploid#Polyploidy_in_plants
Quote:

Polyploid plants tend to be larger and better at flourishing in early succession habitats such as farm fields.




I have never attempted to induce polyploidity with chemicals but in both of my grows one (usually a sativa) turned out polyploid and had an unexpectedly high yield.

Here's an example of a triploid:






Looks like just three buds.  I will have to do more research about breeding polyploidy cannabis as well.


--------------------





Multidisciplinary Association for Psychedelic Studies - Become a member!
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~ Thomas Jefferson ~


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Offlinesupersiege
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Re: Induce polyploidy [Re: FurrowedBrow]
    #260536 - 08/06/09 02:49 PM (4 years, 8 months ago)

i thought that was called a trifoliate, or does that mean the same thing?
nm i found out
Trifoliate = descriptive term for having 3 sets of leaves


Edited by supersiege (08/06/09 03:35 PM)


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Offlinesupersiege
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Re: Induce polyploidy [Re: supersiege]
    #260589 - 08/06/09 03:59 PM (4 years, 8 months ago)

ok so this thread definatly caught my attention and made me do some researching, after scouring dozens of forums and scientific botany sites here are a few interesting things i come across on the subject. The first is a link to Jorge Cervantes rx book inquiring about the chemical
http://books.google.com/books?id=WRNNS2TUB3YC&pg=PA84&lpg=PA84&dq=polyploid+marijuana+jorge+cervantes&source=bl&ots=JKmCHkQySV&sig=sDlkYAyXhjSR0X53OD_66vJyqS8&hl=en&ei=7DB7SrSlEZTCMIu60foC&sa=X&oi=book_result&ct=result&resnum=1#v=onepage&q=&f=false

also i remember in a back issue of high times i have a reader wrote in about his trifoliate plant, jorges response was that they are usually genetically weak.

Here are a couple random excerpts from forums i found as well dealing with polyploid marijuana

Normal cannabis is diploid, meaning that it has two sets of chromosomes - one from each parent - in each cell.

Polyploid cannabis is sometimes referred to in breeding literature, and used to be thought of as a bit of a Holy Grail amongst plants - I guess due to the hope that interesting new mutations might occur when polyploid cannabis was flowered or used in breeding (or maybe just the idea that 'more genes iz better').
AFAIK, there were never any interesting results from breeding with polyploid plants (though the legends do persist).

NB - It's important to note that whorled phylotaxy (plants growing three or four leaves/branches per internode) is not the same as polyploidy. The two terms are often confused - such as in the original version of this post.
One reason for the confusion of the two ideas is that both refer to a 'doubling' of cannabis plants - genes in one case, foliage in the other. It's also interesting that both polyploidy and whorled phyllotaxy were pursued as big breeding breakthroughs, and both came to nothing AFAIK.


I've not heard the term tetraploid before, but since the 'tetra' refers to 'four', I'm guessing it's an update on the less-specific 'polyploid'.
I don't know of any seed strains that have been bred to produce polyploid plants.
From the fact that it was thought of as a desired trait in breeding circles, but was never successfully introduced into a seed line suggests that it's either a random mutation that's not reliably passed on to offspring, or that it proved to be not such a desirable trait after all.

What makes you think you have a tetraploid? Just because a plant exhibits tri-foliate or tetra-foliate leaf patterns, also known as whorled phylotoxy, I beleive, does not mean it is a polyploid. Polyploidism can only be confirmed through genetic mapping.


Here is a nice little read on polyploidism in cannabis, notice some key points I have highlighted for you.

Marijuana Botany, Robert Connel Clark, pub. 1981
Polyploidy

Polyploidy is the condition of multiple sets of chromosomes within one cell. Cannabis has 20 chromosomes in the vegetative diploid (2n) condition. Triploid (3n) and tetraploid (4n) individuals have three or four sets of chromosomes and are termed polyploids. It is believed that the haploid condition of 10 chromosomes was likely derived by reduction from a higher (polyploid) ancestral number (Lewis, W. H. 1980). Polyploidy 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 dip bids (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, Anomalies 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 polyploidy 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. Coichicine 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.

And here is a link to the original thread on ICMag where this clipping was taken from.
http://www.icmag.com/ic/showthread.p...ight=polyploid

and one last clip out of the growers bible on the subject

http://books.google.com/books?id=fERzFsZhdxYC&pg=PA458&lpg=PA458&dq=polyploid+marijuana+jorge+cervantes&source=bl&ots=t0RAcvPGVr&sig=WP72dcbIkfrhKMqoB5cu-lCOUjY&hl=en&ei=czV7SuDRIpK4NcSBzdkC&sa=X&oi=book_result&ct=result&resnum=4#v=onepage&q=&f=false


Edited by supersiege (08/06/09 04:06 PM)


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Offlinesnufkin
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Re: Induce polyploidy [Re: supersiege]
    #260596 - 08/06/09 04:08 PM (4 years, 8 months ago)

Thank you for clearing up the confusion. I stand corrected, my pictured plant is not triploid, but it is trifoliate.


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Grows: 1 2


Edited by snufkin (08/06/09 04:09 PM)


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Re: Induce polyploidy [Re: snufkin]
    #260610 - 08/06/09 04:17 PM (4 years, 8 months ago)

still very interseting, ive been growing for 8 yrs and only come upon one trifoliate and of course it was a male, and it didnt start out with 3 sets it split into 3 branches on the 4th node...but either way from what ive read snufkin you are very lucky to have that lady


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Re: Induce polyploidy [Re: supersiege]
    #260759 - 08/06/09 08:52 PM (4 years, 8 months ago)

>And here is a link to the original thread on ICMag where this clipping was taken from.
http://www.icmag.com/ic/showthread.p...ight=polyploid

that link does not work.  Thanks for sharing that information.  I actually read it all.  Marijuana botany is probably the next book i will buy now.  Very interesting stuff.  Basically, what I have gathered is that it's really not worth it to experiment with this method.  A full-blown scientific study with an actual laboratory would be fucking rad though.  It seems logical that an increase in the chromosomes would lend itself to higher potency chron.


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Re: Induce polyploidy [Re: FurrowedBrow]
    #261019 - 08/07/09 01:45 AM (4 years, 8 months ago)

what if the addition of chromosomes is kind of like evolution and this may be what pot is 250 yrs from now, in one of the 1970's studies it said a polyploid produced more thc but less cannabinoida. Correct me if I'm wrong been awhile since i read some of this stuff but aren't cannabinoid naturally accruing in the body and also help in the processing of THC. So maybe when the world comes to its senses and everyone smokes weed we too will evolve having more natural cannabinoid and be able to process the extra THC from these future plants...i dunno its late and I'm stoned


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