Inducing polyploidy in plants

[Dr.Mantis]

Hi everyone,

Induction of polyploidy in diploid plants is a well established technique in horticulture. Although polyploids can occur naturally in many plants, using microtubule inhibitors offers much more control, and selectivity.

This thread is the start of my experimentation in inducing polyploids in clonal cannabis. My ultimate goal is increasing the volatile organic fractions of the plant, and other secondary metabolites. I don’t care too much about THC (within reason). This is not intended to be a guide, rather documentation of my experiments.

To start this off, I took a clone I don’t particularly care about (GH cheese). Here she is with the first sprouts coming off the diploid plant. The mangled leaves are likely of mixed ploidy levels. As the new shoots progress, I will take cuttings from the largest most uniform shoots. The risk here is polyploid plants tend to be less vigorous to than their diploid parents, so one needs to isolate the polyploidy cells asap. One way to do this is through cloning, and the other is through meristem culturing (trying to avoid that if I can). Ploidy level will be analyzed through stomata density, and root tip analysis.


From here I will refine my techniques and apply it to other clones I care about more. Stay tuned!

 

[Dime]

What specifically are you using to induce polyploid ,is this an attempt to eventually get polyploid offspring?

 

[Dr.Mantis]

What specifically are you using to induce polyploid ,is this an attempt to eventually get polyploid offspring?

Hey,

I’m using Oryzalin at 20uM and 1000ppm of tween20 to keep it in suspension. I think you can use colchicine too, but it’s more toxic to mammals and not as effective in plants. The goal is to produce tetraploid plants, but there will likely me some mixed ploidy species in the mix. Once I have a tetraploid plant, I plan on selfing it to try and produce tetraploid seeds.

 

[Dime]

Yes I heard it was poisonous and can get absorbed the skin. I remember talk of seed soaking in Colchicine most would die but the few remaining showed freaks and I don't recall if the mutations were passed on in the offsprings.. I also read Xrays used to disturb the chromosomes. The Orzalin is also used as an herbicide and has caused organ changes in test animals and is possibly a carcinogen. Be careful.

 

[troutman]

If a tetraploid crosses with a diploid the results will be a triploid and infertile.

Seedless fruits are triploids made by that actual cross.

 

[Dr.Mantis]

Yes I heard it was poisonous and can get absorbed the skin. I remember talk of seed soaking in Colchicine most would die but the few remaining showed freaks and I don't recall if the mutations were passed on in the offsprings.. I also read Xrays used to disturb the chromosomes. The Orzalin is also used as an herbicide and has caused organ changes in test animals and is possibly a carcinogen. Be careful.

Yeah seed soaking is one method, but the viability is usually low. With node treatment you get many chances, and the ability to compare the polyploid to its diploid mom. Yeah also worth noting oryzalin is toxic, but not as toxic to mammals as colchicine, it is used as a pre emergent herbicide in much higher concentrations. Plus I am only using about 100mg of a 20uM solution to treat each node, so we are talking millionths of a gram at this point. However, should not smoke or consume treated plant tissue as a best practice, and ppe is mandatory when handling it.

Triploids seem cool, but I don’t want sterility in the event I want to explore a line for multiple generations. Iirc there is a company making triploid cbd lines in Oregon via the colchicine method.

 

[troutman]

Somebody, may say your polyploids are GMO cause they have more genes than normal.

 

[chilliwilli]

Don't forget the non reproductive lines that could be created.

 

[St. Phatty]

What about inducing genetic variations using radiation ?

Every smoke detector has a VERY potent radiation source in it, 0.1 micrograms of Americium.

Americium-241 - Wikipedia

en.wikipedia.org

But Americium decays MOSTLY by spitting out a Helium Nucleus (alpha particle).

I suspect that X-rays would be better, but they are a little harder to come by.

You can imitate what they do in PET scans, shoot a beam of electrons at a metal target, the heavier metal the better.

The circuitry in an old TV set can be very useful. The piece of steel that you shoot the beam of electrons at doesn't need to be fancy, but it does have to be precise.

One way to subject seeds to X-rays is to send them through the mail, but then you don't control the dose.

 

[Dr.Mantis]

Somebody, may say your polyploids are GMO cause they have more genes than normal.

Yeah, it’s not a GMO because polyploidy does occur naturally ( at much lower rates). However, I could certainly see how people might think they are.

 

[Dr.Mantis]

What about inducing genetic variations using radiation ?

Every smoke detector has a VERY potent radiation source in it, 0.1 micrograms of Americium.

Americium-241 - Wikipedia

en.wikipedia.org

But Americium decays MOSTLY by spitting out a Helium Nucleus (alpha particle).

I suspect that X-rays would be better, but they are a little harder to come by.

You can imitate what they do in PET scans, shoot a beam of electrons at a metal target, the heavier metal the better.

The circuitry in an old TV set can be very useful. The piece of steel that you shoot the beam of electrons at doesn't need to be fancy, but it does have to be precise.

One way to subject seeds to X-rays is to send them through the mail, but then you don't control the dose.

I don’t think radiation would work. For polyploid induction you want tubule inhibitors. Basically, as the cell is naturally dividing through mitosis it doubles it’s copies of genetics material. Then, the tubules pull the copies to each side, and the cell splits in two. Inhibitors stop this process, so you get a cell that has extra dna and no way to properly split. Some of the cells die, and others go on to reproduce with extra copies of their genomes.

Ionizing radiation would probably sterilize the seeds in large doses, or induce random mutations in the genome in smaller doses.

 

[troutman]

Yeah, it’s not a GMO because polyploidy does occur naturally ( at much lower rates). However, I could certainly see how people might think they are.

There's actually a rare tetraploid Cannabis Landrace that could be interesting if only somebody could get seeds.

Abnormal Meiosis in Tetraploid (4x) Cannabis sativa (L.) from Lahaul-Spiti (Cold Desert Higher Altitude Himalayas)-A Neglected But Important Herb

https://www.eresearchco.com/articles/abnormal-meiosis-in-tetraploid-4x-cannabis-sativa-l-from-lahaulspiti-cold-desert-higher-altitude-himalayasa-neglected-bu.pdf

​

 

[Dr.Mantis]

There's actually a rare tetraploid Cannabis Landrace that could be interesting if only somebody could get seeds.

Abnormal Meiosis in Tetraploid (4x) Cannabis sativa (L.) from Lahaul-Spiti (Cold Desert Higher Altitude Himalayas)-A Neglected But Important Herb

https://www.eresearchco.com/articles/abnormal-meiosis-in-tetraploid-4x-cannabis-sativa-l-from-lahaulspiti-cold-desert-higher-altitude-himalayasa-neglected-bu.pdf

​

How cool! Thank you for sharing.

 

[St. Phatty]

I don’t think radiation would work. For polyploid induction you want tubule inhibitors. Basically, as the cell is naturally dividing through mitosis it doubles it’s copies of genetics material. Then, the tubules pull the copies to each side, and the cell splits in two. Inhibitors stop this process, so you get a cell that has extra dna and no way to properly split. Some of the cells die, and others go on to reproduce with extra copies of their genomes.

Ionizing radiation would probably sterilize the seeds in large doses, or induce random mutations in the genome in smaller doses.

Did radiation contribute to useful plant evolution during the life of the Earth ?

I'm not saying it's easy to get it just right.

One of the effects of too much ionizing radiation in humans is that the radiation destroys the Companion Chemicals (commonly called enzymes) that help the DNA to repair and duplicate.

Damage the DNA in an useful way, and the DNA may not survive to be useful if the radiation damages/ alters the enzymes it would use to repair itself from the incoming radiation.

I think PATIENCE is one necessary part of the process.

e.g. I have a bunch of green and red pepper seeds, and I have some friends that collect "Hot Glass" - 1930's era glass where they used Uranium to help color it, often an orange color.

The Uranium in those are roughly 0.7% U235, which decays in several different ways, for example like this -
U235 ==> Barium 141 + Krypton 92 + 3 neutrons.

The neutrons are speeding away from the decay event and can contribute additional decay events.

Normally these kinds of reactions also emit photons (X-rays, gammas), neutrinos, etc.

Though the U235 is only 0.7%, it decays about 5 times faster.

I couldn't find a good decay expression for U238. It ends up being lead and sheds radiation in the process.

Anyway, I'd rather handle radioactive materials or high voltage than Colchicine. But only because I've had On the Job training to help me.


re Colchicine - I would be concerned about its Vapor Pressure at different temperatures, and also, can it be in dust form and go airborne ?

 

[Dr.Mantis]

Did radiation contribute to useful plant evolution during the life of the Earth ?

I'm not saying it's easy to get it just right.

One of the effects of too much ionizing radiation in humans is that the radiation destroys the Companion Chemicals (commonly called enzymes) that help the DNA to repair and duplicate.

Damage the DNA in an useful way, and the DNA may not survive to be useful if the radiation damages/ alters the enzymes it would use to repair itself from the incoming radiation.

I think PATIENCE is one necessary part of the process.

e.g. I have a bunch of green and red pepper seeds, and I have some friends that collect "Hot Glass" - 1930's era glass where they used Uranium to help color it, often an orange color.

The Uranium in those are roughly 0.7% U235, which decays in several different ways, for example like this -
U235 ==> Barium 141 + Krypton 92 + 3 neutrons.

The neutrons are speeding away from the decay event and can contribute additional decay events.

Normally these kinds of reactions also emit photons (X-rays, gammas), neutrinos, etc.

Though the U235 is only 0.7%, it decays about 5 times faster.

I couldn't find a good decay expression for U238. It ends up being lead and sheds radiation in the process.

Anyway, I'd rather handle radioactive materials or high voltage than Colchicine. But only because I've had On the Job training to help me.


re Colchicine - I would be concerned about its Vapor Pressure at different temperatures, and also, can it be in dust form and go airborne ?

Yeah, the problem with ionizing radiation is it’s non specific in its mechanism. As you mentioned it can damage all sorts of things besides DNA. It might work, but you would probably need to go through hundreds of seeds/ nodes to find anything. I haven’t looked in the lit for radiation induced polyploids, but perhaps there is a protocol or two out there? Chemical methods are much more specific in their mechanism, so much easier to control and select. I wouldn’t want to work with colchicine too much either, it’s less potent in plants and needs to be used in higher concentrations.

Oryzalin is much more specific to plant culture and less toxic to mammals. Plus at 20uM we are talking about millionths of a gram of material in a single treatment. So, while you still need to be conscientious and use ppe, you are probably going to get more exposure walking on a pre emergent treated lawn.

 

[Dime]

Yeah seed soaking is one method, but the viability is usually low. With node treatment you get many chances, and the ability to compare the polyploid to its diploid mom. Yeah also worth noting oryzalin is toxic, but not as toxic to mammals as colchicine, it is used as a pre emergent herbicide in much higher concentrations. Plus I am only using about 100mg of a 20uM solution to treat each node, so we are talking millionths of a gram at this point. However, should not smoke or consume treated plant tissue as a best practice, and ppe is mandatory when handling it.

Triploids seem cool, but I don’t want sterility in the event I want to explore a line for multiple generations. Iirc there is a company making triploid cbd lines in Oregon via the colchicine method.

H. E. Warmke found increases in drug levels using cochicine and produced triploid and tetraploid but his aim was to reduce potency. Some reseach found that induced polyploid strains used more nutrients,water etc but were much weaker overall in terms of survival and more sensitive to pressures. Marijuana Botany has a small section devoted to induced mutations,it's quite interesting. I don't know anything about it beyond what I have read from Clarke and the people he gives credit too .

 

[Dr.Mantis]

H. E. Warmke found increases in drug levels using cochicine and produced triploid and tetraploid but his aim was to reduce potency. Some reseach found that induced polyploid strains used more nutrients,water etc but were much weaker overall in terms of survival and more sensitive to pressures. Marijuana Botany has a small section devoted to induced mutations,it's quite interesting. I don't know anything about it beyond what I have read from Clarke and the people he gives credit too .

There’s not a ton of new literature on polyploids in cannabis, but there are a few interesting ones. A more recent one supports what you say here. They found tetraploid plants were slower and less successful to root. The plants themselves had larger leaves, high trichrome density , more terpenes, but no statistical difference in overall thc, iirc. I think it would be cool to cross two tetraploid lines to see if you could make some tetraploid seeds with some hybrid vigor. If I can get the tetraploid induction down, I will probably give it a shot.

 

[St. Phatty]

What happens when you use a Trifoliate male as a Pollen source ?

I guess I'm about to find out. I'm taking several clones of the original. Going to place one clone of each in every group of female plants.

"The Literature Says" - IXNAY.

https://www.ehow.com/info_12013003_triploid-plants.html

https://www.agriculturejournals.cz/pdfs/cjg/2016/02/01.pdf

I suspect a more accurate term is simply, Multi-foliate.

My trifoliate male is Hyper-vigorous.

At about the 15th node, it "went straight" - and just started producing a storm of nodes and stems.

The growth is freakish and very impressive.

I like the idea of crossing it with a bunch of females, but maybe the pollen will be inactive.

 

[Dr.Mantis]

What happens when you use a Trifoliate male as a Pollen source ?

I guess I'm about to find out. I'm taking several clones of the original. Going to place one clone of each in every group of female plants.

"The Literature Says" - IXNAY.

https://www.ehow.com/info_12013003_triploid-plants.html

https://www.agriculturejournals.cz/pdfs/cjg/2016/02/01.pdf

I suspect a more accurate term is simply, Multi-foliate.

My trifoliate male is Hyper-vigorous.

At about the 15th node, it "went straight" - and just started producing a storm of nodes and stems.

The growth is freakish and very impressive.

I like the idea of crossing it with a bunch of females, but maybe the pollen will be inactive.

I’ve always wondered what causes the trifoliate leaved plants, I don’t think it’s tri ploidy. AFAIK triploid cannabis pollen is sterile to diploid plants.

I’ve always found the odd looking plants fascinating. I’ve got a crazy looking seedling that I have cloned because she’s so weird.

Let me know if those traits end up in the offspring!

 

[Dr.Mantis]

Update,

All of the treated nodes on the plant successfully pushed. After observing them for a few days I decided to take clones. Although I only want one stable clone, I took a few because I have read tetraploid plants are not as successful at rooting.

Interesting, you can the area where the clones we taken are quit enlarged in the plant. Additionally, the new leaves seem to be much wider than the old ones. Once things root, and leaves stabilize, I will do a stomata density and size comparison to the untreated leaf. I will also do root tip chromosome counting at some point.

Swollen nodes

Wider new leaves

Non treated leaf