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Cannabis in tissue culture


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#1 JanSteen

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Posted 16 January 2017 - 07:36 AM

The title says it all.

 

Exploration and experimenting brings society forward. I think experimenting and doing practical works can aid a lot in understanding organisms, what makes them tick and how they work. I've been working at universities, high schools, colleges, and I see the same things happen everywhere: Theory does not explain practical outcomes. The only thing that can be done in such a case, is live testing.

I could run all kinds of statistical tests, comparison of genetic expressions, dig deep in the literature and so forth. But only parts of that information will stick forever, and most of it will be lost. In Heirlooms grow log, I mentioned tissue culture as an alternative way to keep strains in his library without too much hassle. Heirloom said it would most likely take a lot of effort. I see where that comes from, the learning curve is steep, and people would rather not risk their plants - and hard earned cash - to something with a uncertain outcome.

JanSteen wants to show the mycotopians that tissue culture for plants is nearly as easy as culturing fungi. Heck, it might even be easier, since plants require less specialized media. Especially cannabis, that can grow on virtually anything.

 

Since there's a lot of stuff going on at other fora, and I have no intention - nor motivation - to read that all, I'm just going to do stuff. Blindly based on my base of knowledge about in vitro plant cultures and the cannabis plant as a whole. That means that I'll leave out large amounts of information; if you're interested, go ahead and google, if that leads to an ambiguous answer, than the answer is ambiguous. If it leads to no answer, come and ask me here.

 

I'm trying to start a callus culture, and from there onwards to test a couple of hypothesis/questions:

1. Can you regrow a plant from leaf?

2. How easy is it to grow cannabis in vitro? Are there any practical limitations to household techniques and materials (still air box + autoclave + pipet + tweezers + knives)?

3. Is it possible to regenerate seeds from callus cultures, if so: how?

4. Is it more cost and labor effective to store cultures, instead of cuttings? In other words: what's the cheapest way to keep your genetics?

5. How do autoflowering strains behave in culture ( do they autoflower after a period of time, or do they stay in vegetative mode?)

 

 

I have answers for you. Which are heavily limited due to the fact that I have other stuff to do right now. Busy bees make the best honey, but it can take some time.  ;-) I will add more extensive answers and motivations when I find the time.

1. Yes.

2. I will explain that in this topic. It will be the main storyline here.

3. Yes, I'm going to do that. More information will follow whenever I start doing that.

4. Yes. If you take into account that a callus culture needs medium and a vessel, and a cutting needs medium, a vessel, light, water, temperature and ventilation.. I would say that keeping a 2mL culture tube in the fridge is less expensive than keeping a 24 watts CFL bulb running for 18 hours a day and having to water the plant every week.

5. No answer yet. But it could provide insight in what makes autoflower flower auto.

 

So, I have a lot of questions to be answered beyond what I've written down. This topic will be on the down low until I finish some personal things first, but it's good to have a start.

 

What do I have in culture right now?

Callus: Cannabis indica hybrid called Punisher #2 (P#2). Stage: Transformation.

Cuttings/Shoot culture: Same strain of the same mother. Stage: Sterile shoot culture established.

Seed culture: Autoflower P#2 pink x White Rihanna #2. Stage: Germination in vitro.

 

Pictures: will come soon. I just don't like the fact that I have to enable nearly every script in my browser to upload pictures, I'm quite fond of my privacy. That means that uploading something forces me to manually break down barriers I have built into my browser. If there is a workaround, I would love to hear it.

 

Anyhow, that's it for now.

See ya!


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#2 Heirloom

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Posted 16 January 2017 - 11:25 AM

really looking forward to seeing this.



#3 Opalguy

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Posted 16 January 2017 - 01:40 PM

I tried this with phaleonopsis orchids......a while ago. I never really had any success. I had no-one to ask questions of, the internet wasn't a thing yet, and books really did the topic no justice. Really looking forward to this. 


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#4 JanSteen

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Posted 16 January 2017 - 03:57 PM

Orchids do very well on plain coconut water with 1,5% agar added to it. It contains all the right hormones, and together with some activated charcoal, it should work just fine.

I never tried orchids though. Well.. I did, but ebay sellers ripped me off and kept selling me seeds of some kind of giant grass species. That was an awfully funny joke, but at the same time a poor waste of effort and money.

 

Oh well, let's just get this thing started.

 

General things

 

I will use the metric system. If you want to do conversions, go ahead and try to find a erlenmeyer flask with pints and gallons noted on it. Lab work requires math, if you lack that skill, go to wolframalpha and let it do the work for you. I use it to check my calculations on a daily basis - better to be safe than sorry.

 

Every medium I use has some fixed components/ingredients. My medium contains at least the following ingredients unless specified otherwise:

- MS salts with vitamins (66% strength, available from a lot of places, I got mine from a biochemistry supplier) -> basic nutrient mixture with added vitamins

- PPM (plant preservative mixture, you can find this on ebay or at a tissue culture webstore) -> Antibiotic against most - but surely not all!!!!!! - contams.

- 1.5% - 2% agar agar (99% pure, ebay, grocery store) If you can get your hands on some Daishin Agar, that's better, but not necessary.

 

- Every part of plant is taken with a sharp, clean, sterilized razorblade. Pinching with fingernails is savage in this in vitro world and will be punished by legions of contams.

- Every contamination - no matter how small - is contained (since it became antibiotic resistant!) and autoclaved before disposal. -> skip this part and your family / housemates / you are in great danger. Don't fuck around with this. Don't. If you can't handle that rule, GTFO!

- Procedures take place in a still air box, I'm wearing nitrile gloves (straight from the box, unwashed or whatever).

- All materials like tweezers, knives, plastics etc. are autoclaved while packaged in tinfoil. This is the same as with shrooms. For the sharp things, I use some tissue paper between the material and foil to keep the stuff from punching holes in the foil.

 

- All media are prepared in unsterile condition, boiled until dissolved in a microwave, and then packed and autoclaved for sterilization. This means that some hormones and organic stuff get lost in the process. Deal with it. I add hormones in 20-45% excess and I calculate that on the go. Over here, I mention the final concentration only, since it would be quite tough for readers to understand otherwise. The math wouldn't make sense. If you have filters with meshes below 45 micron, then you could filter sterilize your hormones. If you decide to do that, use the original concentrations. If not, add 30% to the concentration.

 

Things to have in stock before you even start thinking about tissue culture

 

Hormones

- 6-benzylaminopurine (BAP or 6-BAP) @ 1mg/mL -> dissolved in 1M KOH solution by dripping it on powder untill dissolved (usually it takes around 1 mL) then water is added to make the final concentration. Store solution in the fridge for 3-5 months.

- Naphtalene Acetic Acid (NAA) @ 1mg/mL -> Dissolved in 10% HCl solution by dripping it on the powder until dissolved (usually takes around 2-3mL) then water is added to make the final concentration. Store solution in the fridge for 3-5 months.

- Indole Butyric Acid (IBA) @ 1mg/mL -> Dissolved in Ethanol, added water to make the final concentration. Store solution in the fridge and keep dark(!) for 2 months.

NOTE: NAA and IBA are interchangeable (is that really a word? English keeps amazing me!) that means that one could replace the other. Both do have some different effects, but in the broad sense they do the same.

- 2,4-dichlorophenoxyacetic acid (2,4-D) a herbicide that "orders" cells to start dividing uncontrollably. This stuff causes cancer, it's a thing I keep in mind all the time when handling things. Prepare outdoors or in a safe place with a dust mask and gloves. You'll smell it. Once in solution, it is fairly safe to use if you don't spill. Yes, it's dangerous stuff. But I use it. You could do without, it would just take some more experimenting. I added a dye (food coloring) so that my media containing this stuff, can be recognized from the outside. Use with caution.

 

Liquids, Hard- and software

- A Pipette + tips (ebay has some cheap ones that have uL (micro-litre) amounts, those are the ones you'll need if you want to make anything under 100 samples). Pipetting 200uL with a dropper is possible, but far from accurate. If you are good at using wolframalpha.com or you're good at maths, then do whatever suits you. You could change stock solutions to meet your requirements at any time, I mean, it's not a law or anything.

- Agar Agar (99%) pure. Go for at least 10 grams, otherwise you'll come short. Daishin agar is better, because it solidifies better and doesn't interfere much with nutrients. Regular agar is fine. NEVER use the vanilla flavored stuff.

- Demineralised water (enough to make some batches, I keep a stock of around 5 litres). RO water, ionated water, activated water, whatever, nah. That's NOT going to work. End of story. Demineralised or nothing.

- Plastic/Glass wares -> Petri's, tubes, tissue culture vessels (or just polypropylene/PP5 plastic containers). Keep enough in stock to do everything a third time in a row. If something needs to be recultured away from a contamination, your work will be gone if you'll have to wait 5 days for the delivery man. Say, you're doing 5 petri's, then keep another 15 in stock just in case.

- Bleach

- Glassware for soaking things, Glassware to prepare media.

- Tweezers, knives, handy metalworks

- Ethanol / IPA / High percentage alcohol but below 90% Vol. Otherwise the ethanol can't do it's job. It needs water for transportation. You could always dilute high vol. alcohols of course.

- Transparent wrapping foil on the roll. Cut these in 3cm rolls with a sharp knife (do this while using your brain, you'll cut yourself otherwise, no really. It just takes a few minutes to do this tedious task but it you'll be rewarded if you take that time). Otherwise, use the expensive Parafilm. I never used it for anything in my home lab, since the stuff costs an arm and a leg and I'd use a roll within a week.

 

Hormones + pipettes + other materials will cost around 100 bucks to get started from scratch. The pipette will last a lifetime, the hormones probably too if you buy 10 grams of the stuff, the other materials like glasswork as well. Other things are plain throw-away materials. PP(5) plastics can be autoclaved and re-used up to 40 times. Don't throw those out immediately if something goes wrong. Check if it can handle re-use, because that could save you a looooot of money.

Cost reduction comes in numbers in this case. Also, if you've been working with fungi, some of this stuff might already be in your closet tucked away somewhere. I suggest you run the dishwasher and clean that stuff up to use it. It would be a waste to buy thing you already have.

 

Nutrients

- MS salts with vitamins -> that stuff is expensive, yes. It is the main stuff used in literature, and I recommend using it for ease's sake. It makes things easier to understand, easier to reproduce protocols and do the awesome stuff others do.

- If you want to go experimental, you could use coconut water, liquid nutrients, solvable nutrients, whatever, but this complicates things since you'll need to calculate concentrations yourself. I cannot guarantee anything. Every brand has different concentrations, and therefore different results. Vitamins can come from multivitamin pills, or vitamin B pills. Use a quarter pill per litre at most.

- Cannabis can do a while without nutrients, but not for long. A week or so at most.

 

 

That's all I am giving you guys for today. Next up is sterilisation, but I think I have talked about that in the botanist files V1. In the episode after that, I'll explain more about the procedure.

After that, it's just a waiting + updating results game.

Good to have some readers already, I hope you'll enjoy the show!


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#5 HankoDelicious

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Posted 17 January 2017 - 01:29 AM

This is very interesting! I know nothing of invitro plant culture, but this has sparked some thoughts in me! 

 

Thanks :) 


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#6 fungi2bwith

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Posted 17 January 2017 - 01:48 AM

I had a friend that was a Dr. in botany and he did kitchen countertop tissue culture.....I don't recall if he ever did cannabis, I do know he was working with hostas.

 

Anyways, here's a link to a site that sells TC kits..... http://www.kitchencu...t.com/home.aspx


Edited by fungi2bwith, 17 January 2017 - 01:49 AM.

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#7 JanSteen

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Posted 17 January 2017 - 03:26 PM

Well in vitro culture is nothing more than the in vitro stuff we do with fungi when taking rhizomes. There's not much more to it. If you do the media right, the only thing that can go wrong is the sterile procedure.

We grow plants to a point where we want to reproduce the stuff on a larger scale (or just to keep strains in our possession).

 

Media formulations for cannabis (1,5% agar, pH lowered with 2 drops of kitchen vinegar per 250mL to around pH 6):

  • Rooting: 25% MS + NAA/IBA @ 0.6mg/L
  • Vegetative growth: 66% MS + NAA/IBA @ 0.05mg/L + BAP @ 0.4mg/L
  • Stretching growth: 66% MS + NAA/IBA @ 0.5mg/L (Note the difference in MS; more nitrogen prevents root growth to some extent)
  • Sidebranching growth: 66% MS + BAP @ 0.6mg/L
  • Callus growth: 66% MS + 2,4D @ 0.05mg/L + BAP @ 0.3 mg/L + IBA @ 0.08mg/L
  • Add glucose/sucrose/fructose to around 20 grams/L. Table sugar works, but these sugary nitrogen sources are heaven for contams. You could skip this if you'd like. Light malt extract works as well at the same concentrations.

 

Indica hybrids with over 60% of phenotypical characteristics seem to behave better at -10% MS in every medium.

 

I want to call the following text a sterilization protocol, but to be honest, it's not going to be 100% sterile. It's a disinfection.

  • Decide on what you want to take; a cutting with a stem, a shoot (no stem), a piece of stem, a root, whatever tissue you'd like, as long as it's fresh and growing, it should behave the same as any other piece of tissue.
  • Prepare a 10% bleach solution and add a few drops of dishwasher soap to break surface tension
  • Prepare sterilized tap water and add a drop of PPM
  • Media is already prepared following the instructions above
  • Make some sterilized tissue paper by wrapping it in tin foil and autoclave it

 

  1. Cut your plant piece
  2. Give it a tap water wash for 5 minutes
  3. place plant parts inside the bleach solution for 45 seconds up until 3 minutes, depending on the age of the tissue and it's ability to regenerate after such a beating. This takes practice. There is no other way than to just try it out; I found major differences in the same plant with the identical procedure
  4. Then place the plant part inside the sterile water with PPM, keep it there for 4 minutes
  5. Pad the plant part dry on the tissue paper
  6. Place in solidified medium
  7. Close container
  8. Place in shaded conditions, 25 degrees C. Lights: 25Watts CFL bulb will do just fine for 10 regular (9cm) petri's. Aim for the blue spectrum (was that 3000K?) or 'warm white' (6500K?)
  9. Growth in any way should occur within 2 weeks.
  10. If not, check for contams, recheck procedure and make adaptations. Check for contams anyway, they hide pretty well. If after 4 weeks nothing contamed, consider your culture sterile.

Note: Plants will die, you will fail. That's why we write stuff down. If things fail, recheck your procedure. Every part of this process can be optimized if you are willing to try a few times. For that you'll need notes and patience. Luckily cannabis is a very forgiving plant and a 5 noded branch that would yield 2 cuttings, could yield over 20 cultures.

 

Common problems:

- plants turn brown and die -> too harsh disinfection or contams, too much nutrients

- Plants have weird growth -> recheck and recalculate hormone concentrations OR check FAE (which is necessary sometimes!)

 

 

That's it for now!


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#8 JanSteen

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Posted 17 January 2017 - 03:27 PM

Yeah, I don't know what happened there. Something is wrong with the editor. I'm not going to rewrite 45 minutes of text.



#9 Heirloom

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Posted 17 January 2017 - 05:40 PM

Stuff happens ,has to many of us, maybe the internet connection?

I hope you don't mind these pages in photo.

Attached Thumbnails

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#10 JanSteen

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Posted 18 January 2017 - 11:25 AM

I don't mind it at all, but I have to reject the statement that NAA inhibits root formation. The way it's written down, leaves me to interpret - remember, English is not my native language - that they're saying it does that.

It inhibits IBA's function a little, thanks to molecular interference and signal dampening (too many hormones have the opposite of the desired effect, because the organism has feedback loops saying "too many signals, does not compute, ignore signals"). If you take 40 grams of cracker dry shrooms, and you eat another 5 grams, those extra grams aren't making a difference. Well, maybe it does. I have never spoken somebody heroic enough to try it.

 

NAA showed perfectly fine rooting in my cases. But then again, cannabis roots just as well with hormones as without. The time difference w/hormones and without for rooting is about 3-7 days. If it's speed somebody aims for, this is the wrong page. Cuttings would be faster anyway, but it's about culture this time.

There's nothing more I can say about it.

 

Damn, by the way. Did I really say sugar is a nitrogen source? How, why?! I meant carbon source.


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#11 Heirloom

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Posted 18 January 2017 - 03:51 PM

I also just lost a huge post.

Ok The info I posted was only meant to entertain while you regrouped.

The only info I posted was from decades ago and was complicated. With no direct directions to success.

I am glad you will post direct instructions for micropropagation and show us how to.

Your thread will finally bring micropropagation into the collective consciousness of cannabis growers, to be spread .

 



#12 JanSteen

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Posted 19 January 2017 - 12:58 PM

It's a good and informative post, don't get me wrong! The more information, the merrier. Since micropropagation can be done in a gazillion ways, there is no absolute truth. I'm fine with all input we can get, since I think there are so many ways to Rome, it would be wise to just be able to pick a few and try them out. As for the lost post, it seems to be back again after reloading the page. All I got were a lot of error messages and the only clear thing was that it has to do something with Hippie3 or someting. It showed all over the screen. Can you guys see the media formulations etc. in post #7? If so, then nothing seems lost. Next week I'll have a week off from studies, work and other things, so I'll be doing some more culturing. I have undifferentiated tissue growing on the leaf cutting, but we'll have to wait a bit more until we can see if there's shoots forming, or only roots. Does anyone know a free, privacy respecting, photo hosting website? It would help a lot if I can upload projects and pictorials in a way that doesn't take 2 hours. Mycotopia's way of uploading just doesn't work for me, and officially cannabis is still illegal in my country. So what I'm doing, and the information I provide, could get me jailed if I expose myself too much.
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#13 TVCasualty

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Posted 20 January 2017 - 05:55 PM

Cool!

 

This is info I've needed for a while (ratios specific to cannabis) but haven't had time to get around to researching.

 

I'm sitting on some very special seeds that are now too old to sprout but are still probably alive, so I figured tissue culture would be the way to revive the strain that I collected in Hawaii ~20 yrs. ago. Best smoke I ever had before or since; it smells like Christmas (both the buds themselves and when smoking 'em). It had the fattest leaflets I've ever seen on a cannabis plant, too (some of them overlapped by nearly an inch). The closest strains to it are the old-school Kona Gold and Puna Butter.

 

It was supposedly an original strain of Maui Wowie and I scored four seeds from a local, then when I got home I grew them and got 3 females and one male (perfect!) so I seeded the hell out of the females and still have a bunch of 'em.

 

Best part about tissue culturing from a fertilized seed is that (if I understand the process correctly) I'd end up with two strains (the mother plant's DNA from the inner seed coat as well as the DNA from the fertilized embryo).

 

I sure hope I can get to this project soon as time is probably of the essence...


Edited by TVCasualty, 20 January 2017 - 05:57 PM.


#14 Heirloom

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Posted 20 January 2017 - 11:08 PM

TV it would be great if you could save the Christmas bud been a long time since I had any and would be nice to know that it could be around for future generations to enjoy.


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#15 JanSteen

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Posted 21 January 2017 - 11:14 AM

I don't know if the inner seed coat is viable in culture. The coat loses viability not long after the ripening of the seed. It's a transparent husk with only a few layers of cells which might not divide after the seed has become detached from the plant. If it's even possible, I would think there's a very, very small success rate.
But then again, if you don't try it, you'll never know.


0.5mg/L BAP showed too much callus formation after 20 days, cells are dividing rapidly but lost all differentiation. This happens at the stem, as well as the leafs coming from the nodes. Next I'll try 0.25mg/L to see if there's a better response.
I'm just running trial and errors, in no way the media formulations are factual. They're experimental dosages I am going to try and see the results.

For difficult seeds, I would suggest a hormone called Gibberillic acid (GA3) at very low dosage (0.05mg/L) to break dormancy. However, a good time in the freezer seems to have the same effect.
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#16 TVCasualty

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Posted 21 January 2017 - 12:23 PM

Not the best news, but here's to hoping.



#17 Heirloom

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Posted 21 January 2017 - 01:53 PM

Maybe not the best news but I have a crazy idea. We got a University nearby they use a 22 cal shell to shoot genes into a gel
that has the targeted growth , like another plant in vitro.

I would remove the lead bullet and powder from a 22 shell. They use gold so I would try 24 carat gold leaf  rubbed on the old genetic material and then  shot into the target - this can implant the genes from one plant into another. This is then grown out to a plant, and then rated for desirability. I believe many would need to be done. Though in an hour time frame a whole bunch could be done.

There are variations of this I bet and we know it as genetic modification .

If you can get even a root tip you might reach your noble goal.

edit- I learned of this from a science student about 30 years ago. When his plans to implant cannabis genes into lawn grass he was shut down. His goal to make a high grass than you mow your lawn and extract the THC . He was shut down because "loose lips skink ships" He told 2 people who words went up the grapevine.

 This might very well be possible = I mean to save old varieties.


 


Edited by Heirloom Spores, 21 January 2017 - 02:16 PM.

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#18 TVCasualty

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Posted 21 January 2017 - 02:14 PM

Ah yes, the "gene gun." I think it was invented at Auburn University and uses (or originally used) gold nanoparticles to transfer the genetic material; larger particles would probably shred the cell walls too much, killing them outright. I've wanted one for years; ever since I heard of them I've been dreaming about shooting the genes responsible for THC production into kudzu. :weedpoke:

 

And it just figures that a novel form of biotechnology developed in Alabama would involve firearms, lol.


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#19 Heirloom

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Posted 21 January 2017 - 03:32 PM

You know more than I do on this subject. Good luck buying a gene gun  maybe e- bay  LOL.


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#20 Heirloom

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Posted 21 January 2017 - 04:00 PM

Does anyone think nano particles of gold could be made by using 2 gold electrodes in distilled water, running  like 9 -12 volts DC
through the water? Use the electricity to peal off molecules of gold or maybe atoms into the distilled water. ?






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