# Tissue culture and cannabis?



## MikeGanja (Dec 27, 2014)

Hi,
I found some information on a cloning method called "tissue culture" or "tissue cloning". It has been around for some years and it looks like a possible alternative to normal cloning:






I am just curious why we don´t hear much from this method in cannabis growing. Is it because it doesn´t work or is it because normal cloning works perfect with better results?
Do you know of anybody using the tissue culture method for cloning cannabis?


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## mudballs (Dec 27, 2014)

cost is astronomical for normal growers. it's not as simple as getting some agar plates and going to town on em.


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## MikeGanja (Dec 28, 2014)

Ok! When something sounds to good to be true...


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## mudballs (Dec 28, 2014)

add to the fact that going from cell culture to something you can put in the soil is like 6 months.


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## MikeGanja (Dec 28, 2014)

6 month? Monstergardens.com did not mention the timeline in this product:

https://www.monstergardens.com/Propagation-and-Cloning/Tissue-Culture-Kits-And-Accessories/Plant-Tissue-Culture-Microclone-Kit

They did mention:

*Why Micropropagation?*
- Replaces Mother Plants
- No Bugs, Pest, or Care required
- Greater multiplication
- Super starts
- Vigorous, bushy plantlets
- Less space and light (eliminate your mother room)
- Unlock hidden genetics
- Jars make transportation easy and shippable
- Easy and fun


I liked the idea of eliminating the mother room and get rid of bugs but 6 month is a very long time. I guess that I after a full cycle would have plants in all stages. But doing that would mean that I still have all the problems with bugs. The only benefit would be no motherplant.

Your information have saved me $ 190.


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## mudballs (Dec 28, 2014)

looks like they've come a long way from lab to diy since i looked into it. only thing that makes that remotely attractive is the 'unlocking genetics' issue and i'm not sold on that without getting back into researching it. i thought it was always a duplicate like a clone. hey if you have the expendable income go for it. but i think you're beginning to see why its got a large price tag. like any hobby that kit is just to get your foot in the door.


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## Native Humboldt (Dec 30, 2014)

I experimented with tissue culture a couple of years ago and belong to a forum on the subject. Its not very expensive to get set up the forum will supply you with everything except the container and charcoal filter for around a hundred bucks. The people on the forum are so intelligent on the subject it's amazing and there are no rude comments or assholes on the site. I used a 20 gallon fish tank that I bought off CL for 10 bucks. I was able to culture roses, blueberry's , and orchards but I'll be dammed if I could find the right mix of vitamins to get the MMJ to take. The key to tissue culture is a sterile environment and the perfect combo of a vitamin mix to feed the plants while keeping them in a suspended life cycle. Most plants have a formula that is proven to work if you follow the directions to the letter they will tissue culture perfect. I messed around with it for over a year then got frustrated and gave the whole set up away.


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## MikeGanja (Dec 30, 2014)

Mudballs, I agree. It looks like the process probably could be made at home to a somewhat reasonable cost but the kits like the one in the link doesn't guarantee a successful clone with cannabis. I would be more convinced if Bill Graham show us examples with cannabis instead of redwood.

Native Humboldt, great news that I could get the needed equipment for $ 100. It's less then half the price I found so far.. Sorry to hear that you didn't succeed in finding a working formula for cannabis.

Some Asian scientists have published a study on tissue cloning of cannabis. Do you think it can be useful for growers like us? --> http://www.pakbs.org/pjbot/PDFs/41(2)/PJB41(2)603.pdf


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## Native Humboldt (Dec 30, 2014)

Yes I think 


MikeGanja said:


> Mudballs, I agree. It looks like the process probably could be made at home to a somewhat reasonable cost but the kits like the one in the link doesn't guarantee a successful clone with cannabis. I would be more convinced if Bill Graham show us examples with cannabis instead of redwood.
> 
> Native Humboldt, great news that I could get the needed equipment for $ 100. It's less then half the price I found so far.. Sorry to hear that you didn't succeed in finding a working formula for cannabis.
> 
> ...


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## mudballs (Dec 30, 2014)

MikeGanja said:


> Mudballs, I agree. It looks like the process probably could be made at home to a somewhat reasonable cost but the kits like the one in the link doesn't guarantee a successful clone with cannabis. I would be more convinced if Bill Graham show us examples with cannabis instead of redwood.
> 
> Native Humboldt, great news that I could get the needed equipment for $ 100. It's less then half the price I found so far.. Sorry to hear that you didn't succeed in finding a working formula for cannabis.
> 
> Some Asian scientists have published a study on tissue cloning of cannabis. Do you think it can be useful for growers like us? --> http://www.pakbs.org/pjbot/PDFs/41(2)/PJB41(2)603.pdf


i reviewed that and all that paper describes is the most expensive method to root clones.
native humbolt has the most pertinent info to proceed. sterilization and proper vitamins mix. 
i'm gonna send you to this thread
https://www.rollitup.org/t/scarholes-root-pruning-sex-reversal-method-for-fem-seeds.853829/
in this thread scarhole discusses a prominent breeder culturing root cuttings. if you go down that rabbit hole you might find a way of just taking a cutting from a root and growing a plant.imagine how many more clones you could get from a mother? i'm fascinated by it but not in a position to pursue it at the moment.


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## RM3 (Dec 31, 2014)

Wanna get serious ,,,,,,,

http://www.christian-marijuana.org/Downloads/Miscellaneous/TheBiotechnologyOfCannabisSativa.pdf

https://www.dmt-nexus.me/users/cosmicspore/The_Biotechnology_of_Cannabis_sativa_2nd_edition.pdf


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## Uncle Ben (Jan 1, 2015)

15 years ago I dicussed "mericloning" (cloning from meristem tissue) with a few Dutch growers and seedbanks. They weren't interested. It's far easier and profitable to sell and mail seeds.

25 - 30 years ago I focused my orchid purchases on AOS awarded mericlones. Probably 80% of my orchid collection were mericlones, the best of the best.


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## MikeGanja (Jan 1, 2015)

Thank you mudballs and RM3 for great links and documents. I think you have answered my questions on tissue cloning and cannabis. It looks very interesting.

Perhaps not the most economic way to produce clones but I like the idea of having the favourite strains in agar instead of motherplants. And the concept is really fascinating.


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## MikeGanja (Jan 1, 2015)

Uncle Ben said:


> 15 years ago I dicussed "mericloning" (cloning from meristem tissue) with a few Dutch growers and seedbanks. They weren't interested. It's far easier and profitable to sell and mail seeds.
> 
> 25 - 30 years ago I focused my orchid purchases on AOS awarded mericlones. Probably 80% of my orchid collection were mericlones, the best of the best.


I have seen a lot of info on tissue cloning of orchids. Is tissue cloning used in the orchids industry because it's difficult to grow orchids from seed? Culturing roses and orchids must be the most beautiful cover for a grow op.


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## SnapsProvolone (Jan 1, 2015)

http://skunkpharmresearch.com/tissue-culture/


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## Daub Marley (Jan 3, 2015)

Tissue culture is just micro-cloning. It's more time consuming and expensive than regular cloning, but you can produce much more with less stock and you can remove disease and viruses. It really depends on which ones though because some are easy and others require more equipment, knowledge, and experience to remove. You cannot change the degradation of DNA with tissue culture. It will not revitalize the genetics of your old mothers and it will not replace them.


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## Uncle Ben (Jan 4, 2015)

MikeGanja said:


> I have seen a lot of info on tissue cloning of orchids. Is tissue cloning used in the orchids industry because it's difficult to grow orchids from seed? Culturing roses and orchids must be the most beautiful cover for a grow op.


About the same difficulty. With cloning, you get a genetically identical plant, or rather a bunch of them like 50 at a time.


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## Native Humboldt (Jan 4, 2015)

Daub Marley said:


> Tissue culture is just micro-cloning. It's more time consuming and expensive than regular cloning, but you can produce much more with less stock and you can remove disease and viruses. It really depends on which ones though because some are easy and others require more equipment, knowledge, and experience to remove. You cannot change the degradation of DNA with tissue culture. It will not revitalize the genetics of your old mothers and it will not replace them.





Daub Marley said:


> Tissue culture is just micro-cloning. It's more time consuming and expensive than regular cloning, but you can produce much more with less stock and you can remove disease and viruses. It really depends on which ones though because some are easy and others require more equipment, knowledge, and experience to remove. You cannot change the degradation of DNA with tissue culture. It will not revitalize the genetics of your old mothers and it will not replace them.


If I remember correctly tissue culture does restore the plants original characteristics. Its been a couple of years since I have studied tissue culture but I seem to remember reading that. I'm thinking about trying it out again this year just to see if I can get some plants to survive for a few months and then growing them out while documenting the results. Anyway it sounds fun to give it another shot, you guys inspired me I guess....


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## Uncle Ben (Jan 4, 2015)

Native Humboldt said:


> Anyway it sounds fun to give it another shot, you guys inspired me I guess....


You have a $200,000 lab complete with centrifuge, etc?


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## Native Humboldt (Jan 4, 2015)

Uncle Ben said:


> You have a $200,000 lab complete with centrifuge, etc?


No I have a small sterile home set up, I learned from a tissue culture group a couple of years ago. My wife and I have had really good success with roses and blue berries but I just couldn't get the mix right for cannabis. It would survive for a month maybe 6 weeks then the whole dish would die off. I got frustrated after several tries and lent my set up to a friend who has had a little better success than I had but still isn't there yet. I'm not sure where the $200,000 price tag came from, but yes if I needed one and was motivated I would buy it!


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## MikeGanja (Jan 4, 2015)

I need to study the documents in this thread a few more times and perhaps read some literature on basic lab technique/”biotechnology at home” but I will also give it a try. 

In the “The Biotechnology of Cannabis sativa” the author included a list with companies that are selling equipment. Most of the products are sold in small quantities to hobbyists. The most expensive equipment I have found so far is a pressure cooker for autoclaving and a small aquarium to build a sterile environment.

Native Humboldt, did you build a glovebox or a flowhood for your setup?


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## Native Humboldt (Jan 4, 2015)

MikeGanja said:


> I need to study the documents in this thread a few more times and perhaps read some literature on basic lab technique/”biotechnology at home” but I will also give it a try.
> 
> In the “The Biotechnology of Cannabis sativa” the author included a list with companies that are selling equipment. Most of the products are sold in small quantities to hobbyists. The most expensive equipment I have found so far is a pressure cooker for autoclaving and a small aquarium to build a sterile environment.
> 
> Native Humboldt, did you build a glovebox or a flowhood for your setup?


I built a flow-hood equivalent to a class II that is basically a HEPA filter built into a recirculating fan that scrubs the air. I also pulled the air through a crushed carbon filtering system. I wish I would have taken pictures of the set-up. I had the clean room set-up inside my shop in the back corner. I think the room was 5x7 and was constructed with 2x4's with a 10 mil layer of clear poly on both sides, ceiling, and floors. The room had a positive pressure created with the HEPA filtered hood. Inside the room I had several set-ups the most successful was a aquarium with filtered air. That's about it besides always wearing clean cloths and removing shoes before entering the clean room. Good luck with your project...


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## Daub Marley (Jan 4, 2015)

Native Humboldt said:


> If I remember correctly tissue culture does restore the plants original characteristics. Its been a couple of years since I have studied tissue culture but I seem to remember reading that. I'm thinking about trying it out again this year just to see if I can get some plants to survive for a few months and then growing them out while documenting the results. Anyway it sounds fun to give it another shot, you guys inspired me I guess....


Can you explain how that is possible or point me to a reference?


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## Native Humboldt (Jan 4, 2015)

Look it up in the journal of cell biology or just google it. There are several studies on the subject. Like I said its been a while since I have read about the process. In the near future I'll dive back in just too busy right now..


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## Uncle Ben (Jan 4, 2015)

Native Humboldt said:


> No I have a small sterile home set up, I learned from a tissue culture group a couple of years ago. My wife and I have had really good success with roses and blue berries but I just couldn't get the mix right for cannabis. It would survive for a month maybe 6 weeks then the whole dish would die off. I got frustrated after several tries and lent my set up to a friend who has had a little better success than I had but still isn't there yet. I'm not sure where the $200,000 price tag came from, but yes if I needed one and was motivated I would buy it!


With orchids they use a centrifuge which somehow induces asexual reproduction. They keep spinning/stopping and when they have enough plantlets they let them grow until washed out of the nutrient agar and potted up with tweezer into 3" community pots. They are separated after some growth & then individually potted into 2" pots.

Your setup sounds like quite a feat!


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## thump easy (Jan 4, 2015)

ill run a lil down to you its not hard at all i did it with over the counter nutrients and hormones, i got the sett up from one of those box deals they send you the only fucked up part is the information didnt mach so you'd fuck up because the directions on the video say one thing but the chemicals say another he changed it so your not to shure what the fuck the instructions realy say he has this 1989 vidoe poor footage wich would be great if you could understand it but its not cool a recall would have been great but instead i called the guy and i failed i later seen a few documentaries from univercities on the subject on youtube long and dragging but you get hints and   key words like vitamins and hormones one for shoots the other for roots


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## thump easy (Jan 4, 2015)

most important you need a steril air and equipment if you dont got this contamination turns into a percentage of loss i dont care how clean you are you need a lab envirment or contamination is enevitable, and death come with contamination


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## Native Humboldt (Jan 4, 2015)

It would be really nice if 3 or 4 of us put together similar setups and worked together trying different combinations of vitamins along with humidity and temp to achieve the same goal. I'm sure with the plant knowlage and research abilities on this site we could master the process.


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## thump easy (Jan 4, 2015)

but if your technique is on the money you'll have sucess but not totall unless you got everthing on point


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## thump easy (Jan 4, 2015)

thats not the hardest part the hardest part to me is getting them to get an exoscellition or a hardend surface so that they dont go into shock when you pull them out getting them to stableize was the hardeest part for them adapt to the air and climate outside their containers..


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## Native Humboldt (Jan 4, 2015)

thump easy said:


> thats not the hardest part the hardest part to me is getting them to get an exoscellition or a hardend surface so that they dont go into shock when you pull them out getting them to stableize was the hardeest part for them adapt to the air and climate outside their containers..


Your right but we are dealing with a weed that is pretty forgiving I had great luck with the roses so I'm pretty sure my basic process is correct it just needs to be tuned a bit.


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## thump easy (Jan 4, 2015)

i got the vitamins its so easy your gona be like what the fuck its that easy yes i did the work and im not a high school grad im a GED kid lolz its realy easy i gota find the notes ill give you guys the recipe its so fucken easy no more than 50 buck the jars and the lids probley the most expensive part.. or wait the presure cooker was the expensive part.


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## thump easy (Jan 4, 2015)

give me a day or so today is my day off but my day off means tend the garden..


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## Sativied (Jan 5, 2015)

Nice job thump! I look forward to more info. 

I've seen attempts and journals at several forums and they typically end before it becomes interesting.

Some links I bookmarked, can't vouch for the quality, but about cannabis specifically:
http://www.lycaeum.org/mv/anagrams/cannabis_tissue_culture.html
http://www.pakbs.org/pjbot/PDFs/41(2)/PJB41(2)603.pdf



thump easy said:


> getting them to stableize was the hardeest part for them adapt to the air and climate outside their containers..


That's the downside of tissue culture many don't realize. Regular cloning is much and much faster.


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## thump easy (Jan 5, 2015)

Sativied said:


> Nice job thump! I look forward to more info.
> 
> I've seen attempts and journals at several forums and they typically end before it becomes interesting.
> 
> ...


thank

Thanks ya the down side is you need money for the equipment and steril everything air ppm purity is expensive and so is the flo hood and all the parts and pieces pluss the room its realy easy its all mostly flora nova veg for the first part i gota find the math but a few drops will do to that pyres glass agar you can get at a health food store for like 9 bucks its rooting hormones for the roots and shoot hormones shit i gota find that paper but ill give you the links of were i got the parts and pieces.. also shoots was an advanced yellow bottle for node production it has a kangaroo on it shit i havent seen that shit hear in the desert but in Whittier California it sells no its not advanced the one we know its from australia it promotes shoot growth in flower durring the first two weeks of flower its bomb for node prodution..


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## MikeGanja (Jan 5, 2015)

Native Humboldt said:


> It would be really nice if 3 or 4 of us put together similar setups and worked together trying different combinations of vitamins along with humidity and temp to achieve the same goal. I'm sure with the plant knowlage and research abilities on this site we could master the process.


Excellent idea! Sharing information and experiment with different combinations together could save us a lot of time not doing the same mistakes. It could take a while until I have a working sterile setup but I will document and share my experiments on ROI.


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## MikeGanja (Jan 5, 2015)

thump easy: Thank you very much for input and pictures ( and death scene), very inspiring!  Do you grow the plants in soil or in hydro during the last period, when they first are exposed to the environment outside the test tube?


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## thump easy (Jan 6, 2015)

MikeGanja said:


> thump easy: Thank you very much for input and pictures ( and death scene), very inspiring!  Do you grow the plants in soil or in hydro during the last period, when they first are exposed to the environment outside the test tube?


my last try i put them in aroe most died and in coco most died, the way i kept them alive wich was sad was like on or two i cant remember but i put a plastic coke bottle cut with lil holes in them so that they didnt go directly into shock the nutrients in the aroe i just use like aroe clone or aroe ponic chambers is like 200 or less ppms.. i tried and tried and didnt have very good rounds i always work for clinics and pay electricity so i cant skip a beat its got to be a room fool of course with script but tissue culture is very hard without a lab environment, im very smart dont let the spelling get it twisted, i first wash off the agar with ph water just the roots and then introduce them into the cloner with a dome and little holes so the exterior of the plant can harden and acclimatize to the room temp if you let them out bare they will die quick real quick or get sick.. that was as far as i got if i cant fill up the room i cant live eat or drive or anything Edison taxes big time so the success rate is not that great.. but im sure with a sterile environment it can be done with high rates of success.. im sure of it.. im getting offerd to do work like this in the near future but not really interested anymore its to much work.. dont let me ramble on but for the novelist its great fun.. the truth is you do get bigger plants and vigor and if you make it past the first few weeks they become monsters fast..


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## thump easy (Jan 6, 2015)

i used a couple of drops as you could see in the pic with the droper distrilled water agar mixed with a blender of cake blender nutrients spin thier was a small bottled left from the old package called ppm it helps keep your agar steril you could find it on line its a bit pricey but this is what keep you steril most part, a few drops into the steril water and a few drops into water of rooting hormone you want to micro wave it for about five minute move to steril evirments and with clipings already washed for thirty second in bleach water for 30 seconds and thirty seconds in alcohal ya it sounds crazzy but yes this is how you will get rid of all contaminance ... let me start over i gota find the paper work and the right info i just got off work construction rapped it up about 1130 and im tired ill do a real down to the very very incrament of my movements for you guys but hear is the other products now healthy foods agar powder, and and i dont remember the rooting compound i used i always change them around depending if im steril or using benies in my aroe..


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## AlphaPhase (Jan 6, 2015)

it's basically for preserving genetics. If you want 1000 genetics in a small warehouse, tissue culture is where it's at. It works great. I haven't done this myself, but I know people who have. I wouldn't take a tissue culture for something you want to flower next month though, but it does have it's use.


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## Uncle Ben (Jan 6, 2015)

thump easy said:


> thank
> 
> Thanks ya the down side is you need money for the equipment and steril everything air ppm purity is expensive and so is the flo hood and all the parts and pieces pluss the room its realy easy its all mostly flora nova.....


IOW, it's not practical and the success rate is very low with cannabis as opposed to orchids where the success rate is very high. You adapt to whatever methods works best for you regarding cost and being reasonable.


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## thump easy (Jan 21, 2015)

im gona try it at our new place im gona invest in all the steril equipment and just for shits and gigles im gona try to market them lolz who knows it might work as far as genetics go sounds like another project new place almost done


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## eyes13 (Jan 22, 2015)

thump easy said:


> who knows it might work as far as genetics go





AlphaPhase said:


> it's basically for preserving genetics. If you want 1000 genetics in a small warehouse, tissue culture is where it's at. It works great. I haven't done this myself, but I know people who have. I wouldn't take a tissue culture for something you want to flower next month though, but it does have it's use.


Ideally I want an incubator in addition to the same veg space I use now, so just eliminating a dedicated mother space for ~20 strains I'd wish to hold on to. Does that sound like a reasonable application here? Glove box, filter etc are within reach.


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## AlphaPhase (Jan 22, 2015)

eyes13 said:


> Ideally I want an incubator in addition to the same veg space I use now, so just eliminating a dedicated mother space for ~20 strains I'd wish to hold on to. Does that sound like a reasonable application here? Glove box, filter etc are within reach.


That sounds reasonable. I wouldn't expect much success at the first attempts but practice makes perfect. I wish I had experience with the method because I had to cull many plants that I really wish I had kept for future grows but just didn't have the space to keep them around. Like mentioned above, if the room, tools and equipment are surgery kind of sterile, you'll have much better success. Good luck and keep me updated with how it turns out, it's an interesting topic.


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## AlphaPhase (Jan 22, 2015)

One tip I'd like to mention, even if the equipment is sterile.. there still can be contaminants in the air. Lysol the hell out of the area you plan to work in (spray the air, kill airborne pathogens) and work quickly, this will better the success rate, you wouldn't believe the stuff floating in the air that can contaminate things.


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## thump easy (Sep 7, 2015)

i know it sounds crazzy but just spray a branch with silver spray and seed up a branch cover and tie it off you can save those for years wish i could go in depth but im retired!!!!


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## GrowerGoneWild (Sep 8, 2015)

thump easy said:


> i got the vitamins its so easy your gona be like what the fuck its that easy yes i did the work and im not a high school grad im a GED kid lolz its realy easy i gota find the notes ill give you guys the recipe its so fucken easy no more than 50 buck the jars and the lids probley the most expensive part.. or wait the presure cooker was the expensive part.


 Would you mind posting your notes on your mix for the medium?. 

I got a few mixes i'm gonna try, it looks like you've had success, I'm interested in the final agar mix for rooting. 

Any thoughts on the hardening off process?


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## GrowerGoneWild (Sep 8, 2015)

AlphaPhase said:


> One tip I'd like to mention, even if the equipment is sterile.. there still can be contaminants in the air. Lysol the hell out of the area you plan to work in (spray the air, kill airborne pathogens) and work quickly, this will better the success rate, you wouldn't believe the stuff floating in the air that can contaminate things.


I do something similar when working with mushrooms when inoculating jars, Not like I have a laminar flow hood.. yet.


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## GrowerGoneWild (Sep 9, 2015)

Well I was poking around and heres a kit.. 

http://phytotechlab.com/media/downloads/16477/C1850- Hemp (Cannabis) Multiplication Kit.pdf

Somebody posted the PDF of the 2009 Wang formulation for the TC. Its a commercial kit, now I admit its expensive, but it might be worthwhile to experiment with a kit till you get familar with the process.

I just finished a book on Tissue Culture, and I'll have to post that formula here.. I'm just trying to decipher some of the abbrevieations for the the hormones.


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## thump easy (Sep 9, 2015)

GrowerGoneWild said:


> Would you mind posting your notes on your mix for the medium?.
> 
> I got a few mixes i'm gonna try, it looks like you've had success, I'm interested in the final agar mix for rooting.
> 
> Any thoughts on the hardening off process?


well the ppm you gota get it from a chemical lab its real expesive.. that thier alone is worth it if you dont have a steril 99.0 invirement i can if you give me a week or so but the kit i got originaly was the same incraments i only did a few drops of the other stuff lett me look back thier on you tube you have to sit for a few hours to listen to the proffesors in univerities give their lecture on TC listen for the key ingredients and go to the hydro store and look on the lables on the back you'll find the ingredents i gave up because i have dogs and thier in and out all the time most my cultures would eventualy get lost infected with mold spores.... the original kit changed its chemicals and the guy waited for you to call him so you had one last crack at it the beta commerial was pritty fucked up excuse the aunguage.... thow even the instructions didnt mach.. 250 pluss shipping the world is made of gimiks, its realy easy you do need the presure cooker and you do need lysol you need clean equipment and the augar ill see if i can im pritty bizzy person i wont promise you i will but i tell you just youtube a few lectures and its that easy... Dont rely on anyone dude realy fucken easy im a joe smoe if i did it you can do it. but realy in my experience its not worth it... just s1 you genetix and you have a freezer that what i do... but fo the sake of fun and spending 1000$ go for it if you have the money.. It is very fun i do things for fun even if they cost money.. just fo the sake of doing it good man im shure you,ll get it.


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## GrowerGoneWild (Sep 9, 2015)

thump easy said:


> well the ppm you gota get it from a chemical lab its real expesive.. that thier alone is worth it if you dont have a steril 99.0 invirement i can if you give me a week or so but the kit i got originaly was the same incraments i only did a few drops of the other stuff lett me look back thier on you tube you have to sit for a few hours to listen to the proffesors in univerities give their lecture on TC listen for the key ingredients and go to the hydro store and look on the lables on the back you'll find the ingredents i gave up because i have dogs and thier in and out all the time most my cultures would eventualy get lost infected with mold spores.... the original kit changed its chemicals and the guy waited for you to call him so you had one last crack at it the beta commerial was pritty fucked up excuse the aunguage.... thow even the instructions didnt mach.. 250 pluss shipping the world is made of gimiks, its realy easy you do need the presure cooker and you do need lysol you need clean equipment and the augar ill see if i can im pritty bizzy person i wont promise you i will but i tell you just youtube a few lectures and its that easy... Dont rely on anyone dude realy fucken easy im a joe smoe if i did it you can do it. but realy in my experience its not worth it... just s1 you genetix and you have a freezer that what i do... but fo the sake of fun and spending 1000$ go for it if you have the money.. It is very fun i do things for fun even if they cost money.. just fo the sake of doing it good man im shure you,ll get it.


Thats cool, whenever you can. I just wanna take a peek at your formula, I just want a ballpark idea of what I need for the TC. I'm looking at the chinese formula, and thats available in the kit link mentioned above. And theres a formula by Melinda in her book on Micropropagation. 

Making the agar is easy, what goes into the agar is the question. 

I guess I could use S1's to bank the seeds, but I'm looking at it from a different view. For example: Dark Heart Nursery is looking hard at TC. I bet there is a heavy demand on clones thats why they want to use TC.


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## thump easy (Sep 10, 2015)

GrowerGoneWild said:


> Thats cool, whenever you can. I just wanna take a peek at your formula, I just want a ballpark idea of what I need for the TC. I'm looking at the chinese formula, and thats available in the kit link mentioned above. And theres a formula by Melinda in her book on Micropropagation.
> 
> Making the agar is easy, what goes into the agar is the question.
> 
> I guess I could use S1's to bank the seeds, but I'm looking at it from a different view. For example: Dark Heart Nursery is looking hard at TC. I bet there is a heavy demand on clones thats why they want to use TC.


that would be asome but its faster on the money side to clone out... tc is a hole lab and workers and months before you get a well astablished plant that you could sell a patient if i know money and i know the back side of bizzness its not good for them unless they up the price it takes longer to propigate and longer process to root and get shoots and then to aclimate and then turn around and sell the product!!! if someone really got down testest verifired the quility of a strain it be on like donky kong but clone people sercum to greed or sercumstances letting the quility of the clone go to shit or god know what i have never ran into a consistant great clone place its hard!!!!! good luck and may the force be with you on you quest i hope it works out for you ill post what i can find im cleaning house ill try to get you the info..


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## EverythingsHazy (Sep 10, 2015)

Generally, TC works best if:
-You need a massive amount of identical clones. 
-You need to clone a plant that doesn't root well with traditional methods.
-You need to get rid of a plant virus that is harming one of your strains. 
-The plant you want to multiply takes a long time to become mature enough to flower and set seed.

Most cannabis sellers don't fit into those categories, but that doesn't mean it won't benefit some. Do keep in mind, that it is tedious and somewhat difficult (difficulty level varies depending on skill, technique, and the tissue you are trying to culture). You will probably have lots of failures in the beginning, and you just have to learn from them instead of getting discouraged, or ignoring them and making the same errors over and over.


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## GrowerGoneWild (Sep 17, 2015)

EverythingsHazy said:


> Generally, TC works best if:
> -You need a massive amount of identical clones.


Yes, this is my motivation for TC. And i'm trying to exploit a loophole with unrooted cultures not falling under any legal definition of "cuttings". TC could help me out. Colorado has an 8" rule for cuttings.. etc.. 

Another thing I would like to fix is the rather slow time I use to isolate clones to prevent infection. I currently use 2 weeks to make sure the plant is free from infection, pests and stuff. And then it goes to mom.. And that takes time too. I figgure in the same ammount of time I should be able to have at least 20 clones in the same time I'm ready to take clones from a mother plant.


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## GrowerGoneWild (Sep 22, 2015)

Well here is one possible formulation for Tissue Culture for Cannabis. 

I would suggest reading "Plants from Test Tubes" by Holly Scoggins, its a good primer for TC. 
Then read "Marijuana from Test Tubes" by Melinda Davis.

I would suggest reading these books in that order, it will help you to mix the agar plates as well as help with the basic concepts of TC. What light to use, hardening process.. etc.

MS = Murashgie & Skoog
Dont ask me what formulation of MS, there seems to be many variations of it in the Sigma Aldrich catalog for TC. I'll update this when I get things figgured out. And it seems to be a MS version on Amazon oddly enough. 

There are a few versions, of TC in vitro but here's Melinda's Davis formula. 

Verbatim.. (My kindle is on the fritz, Its from notes I scribbled down.)
Stage 1 from cutting / Stage 2 Divisions
1 MS with vitamins
2 Tablespoons sugar (sucrose)
1 ML PPM
.5ml IBA at 1mg/ml
9g/l Agar.
30 g/l Sucrose

Stage 3 Rooting
1 MS with vitamins
2 tablespoons of sugar
1 ML PPM
1 ML IBA 1mg/ml
1 ML TDZ @ .1mg/ml
9g/l Agar
30g/l sucrose
.5G/L Activated Charcoal.

I'll post something about the Chinese work (Wang 2009 Microprop of Hemp.) Be patient, but the formula is commercialy available.. just got to find that link again. 

Now I understand there's quite a few kits out there, I'm trying to see if I can formulate it from Melindas formula, and this also helped me understand that the TC involves "stages". With 3rd being the rooting stage. 

And I'm cheap,  I would think that it would be more economical to make my own...


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## Voidling (Sep 27, 2015)

The reason I want to tissue culture is to have a library backup of mothers. I have lost some wonderful plants. I was even sharing with two friends so we each had cuts and we had a couple where we all lost our cut of. It wouldn't do away with mothers for me because my grow isn't that large. 

That being said I believe a way to reduce mold and bacteria take over, plus improved hardening off would be in a form of tissue culture called
photoautotrophic propagation

I have studied on tc a lot but life took me away from it for a while. Unfortunately for now I have no money for such things, but when I'm done setting up my business I can write up all the equipment as a business expense for cloning other kinds of plants.


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## GrowerGoneWild (Sep 27, 2015)

I dont know how long explants last or if they could be used as a "library". My motivation for TC is to produce large volumes of clones and clone autflower plants. Would it not be easier to store an F1 or S1 seeds of your mothers plants?.

I looked up photoautotropic propagation, it seems to be different from the TC i'm working on, with the MS medium with sucrose. I tried to read the abstract on Photo TC, but I didn't want to pay the 30 bucks to look at it. Care to comment on why you pick that version of TC?

I want to pick Dark Heart Nursery's brains on what version of TC they are using. As of now the only versions I know that are workable are Melinda's and Wang's TC. I'm more or less finishing up the abstract on Wang's TC, And more or less trying to understand MS formulations, I'm kinda stuck on if the optimum formula was MS, with Gamborg vitamins, and I'll still have to find the formula in Sigma Aldritch catalog. 

I'm pretty close to getting my first TC's going.. Mebby in a week I'll buy some medium.


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## Voidling (Sep 27, 2015)

Just waking up so groggy. I'll try to remember to look up the pdf I have Ann's try uploading somewhere.

It uses a sugarless medium so less chance of nasty things taking hold. Second part is I believe there is a better rate of survival in the hardening off stage. I believe it is similar to a method to certain mushroom cultivation where you have air flowing through an aquarium. 

It may be too complicated or expensive for a home setup. I haven't been able to set up yet to try it yet unfortunately.

People learned how to germinate orchids seeds in vitro back in 1900 and have experimented since to perfect it and move forward with micropropagation.

I have read that every Cavendish banana is tissue cultured. These are the bananas on shelves world wide. Not surprising, they were bred to have no functional seeds. I'm not sure about other commercial crops.

Venus fly traps and the like ate popular for tc, at least amongst hobbyists and they seem to have that down part as well.

The lack of study on marijuana is because it and hemp have been illegal for so long. There is the one Chinese paper I know of that tried it with hemp. I forget their success rate. 

There's only a couple of reasons I see it could be worthwhile other than just liking to tinker and experiment.

1) Keep a back up library of many strains. This in case the mother dies. Or in case you are short on room and want to try new strains still, but don't want to lose other strains you'd need to cull to make room.

2) You're running a large commercial grow. This way you don't need tons of identical mothers to keep product consistency. Even this way you can get variations in size I'd think. My understanding on tissue culture is it's rather uniform in growth when everything is consistent. You wouldn't have to root in multiple batches as the mothers degree enough.

3) Better spread of clone only strains, or maybe hard to clone strains. It theoretically would allow shopping if the person on the other end knew what they were doing. Or to this end possibly the practice of artificial seeds will be perfected for marijuana.

I would say I'm surprised the operations in Amsterdam never picked this up for crop uniformity but sadly I'm not. Those seed companies don't even bother stabilizing their lines. Just grab any two "strains" also them together, also a new name to it and ship it right out the door


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## Voidling (Sep 27, 2015)

F1 isn't stable genetics and would leave you pheno hunting for something similar but would never be exact. This is why there's big variation when buying seeds from seedbanks 

F1? Is that femenizing the seeds? Some believe this is not a good practice for various reasons. Some will say not to breed from a feminized seed. I don't know if these people are any smarter than me but it's not an area I Ann interested in researching things like tc. I'm also not sure how much variation you get this way.

As for explants they should go on forever provided a few things. They will have to be removed, divided, and placed in new agar mix periodically. During the time they are open it must be a sterile environment. My understanding of good Petri dishes is they seal completely to keep out contamination in lab cultures.

If you search here for tissue culture you will find a couple of threads. There it's probably a small amount of good information on each. Unfortunately they invariably get somebody talking about how they've done it for decades and it's so easy but never have proof or give out actual information. One went as far as to claim he had an article all about it in the upcoming skunk magazine but as far as I could tell they had no such article the month he said, nor the next. They disappeared and the threads die


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## GrowerGoneWild (Sep 27, 2015)

Voidling said:


> F1 isn't stable genetics and would leave you pheno hunting for something similar but would never be exact. This is why there's big variation when buying seeds from seedbanks
> 
> F1? Is that femenizing the seeds? Some believe this is not a good practice for various reasons. Some will say not to breed from a feminized seed. I don't know if these people are any smarter than me but it's not an area I Ann interested in researching things like tc. I'm also not sure how much variation you get this way.
> 
> ...


No I'm talking about S1 a selfed female to limit varation. F1's could be stable if the result was a homozygous allele.
Perhaps it doesn't even matter, it would be ideal to grow out each one to pick desired traits. As much as I would like to keep certian phenotype expressions, I have to realize that cannabis hybrids will continue to evolve, what was awesome yesterday can be plain vanilla today.. 

Yeah I'm thinking it could be possible to "continue" the culture, vs a mom in a bucket scenario. If one was to have proper equipment like a laminar flow hood.. I could see this as possible, It just comes down to economics, Its relatively low cost and simple to maintain a mom. 

Yeah I dont really care about RIU contributions, the overall quality has gone down, I'm more of an ICmag person myself, I'd rather spend time looking an actual abstract than going through the threads here. I think I post stuff here mainly as a way to think out loud.


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## GrowerGoneWild (Sep 27, 2015)

Voidling said:


> Just waking up so groggy. I'll try to remember to look up the pdf I have Ann's try uploading somewhere.
> 
> It uses a sugarless medium so less chance of nasty things taking hold. Second part is I believe there is a better rate of survival in the hardening off stage. I believe it is similar to a method to certain mushroom cultivation where you have air flowing through an aquarium.
> 
> ...


Meh, I've run sugarless mediums like in the case of psylosybin cultivation, you can have contamination take hold if the medium has sugar in it or not. It might decrease the likelyhood of infection, so point taken.

Actually the method is not like mushroom cultivation, the byproduct of fungi is CO2, and it does not take much airflow, in fact on the Brown Rice Flour (BRF) cakes I make I do not expose them to circulating air until a fruiting body shows. 

So back to "photoautotropic propagation," the idea is that CO2, is the food versus Sucrose in the agar. If we look at Chieri Kubota's work she says that in vitro propagation is slow due to the limited ammount of co2 in vitro. And I somewhat agree after looking at the time it takes from Stage 1 to plant hardening in Wang's or Scroggins work on in vitro cultivation. Off the top of my head from explant to hardened plant takes somewhere around 6 weeks.

Well at the very least we could flood a laminar booth with a high level of co2 when inserting explants into the desired medium. In Kubota's work it looks like there is more mass vs regular explants. And needless to say co2 we know is a basic building block of mass. I'm now sure how I would implement this and mantain a sterile culture at the same time flowing co2 over explants in vitro, not to mention the cost of concentrated co2, as in a compressed cylinder might push me away from going this route.

The abstract you mention is wang's 2009 study on the micropropagation of hemp... Cannabis Sativa L. Same thing if you ask me as the typical cannabis hybrids. The success rate after Stage 3 to hardened plants is 99%. 

Well I'm researching this now, I think I'm going to go commercial, and provide clones to commercial grow ops.. I'm looking at pushing clones out in numbers like 200-500 clones a month. 

At the very least I've learned a bit, from Kubota's work, I could use the co2 to optimize growth in my explants.  I'll continue to at least look into the subject.

Like.. good stuff man...


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## Voidling (Sep 27, 2015)

Ah yeah selfed female is a feminized seed.

I started at icmag, came over here for a single thread that was a community. Was sent some great genetics from friends I made. Unfortunately the thread died. Now I don't do much on the forums, and usually nothing outside of the diy led. 

I had bought spore syringes but then the large pressure cooker I was going to borrow supposedly suddenly wouldn't hold pressure the night before I was to pick it up. So the syringes went to waste. Never have gone forward. I need to, seriously need to try micro dose for serious depression. 

Maybe need to vent off the mushroom co2 to the explants, or alcohol making. If only that simple.

Yeah I assumed the recipe that works for hemp would work for marijuana. 

Let me know how it goes, I'm interested


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## vitamin_green_inc (Sep 27, 2015)

Voidling said:


> Ah yeah selfed female is a feminized seed.
> 
> I started at icmag, came over here for a single thread that was a community. Was sent some great genetics from friends I made. Unfortunately the thread died. Now I don't do much on the forums, and usually nothing outside of the diy led.
> 
> ...


Don't need a pressure cooker


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## GrowerGoneWild (Sep 27, 2015)

vitamin_green_inc said:


> Don't need a pressure cooker


Heh, yeah you beat me to it.. you dont need a PC, I've found 90 min of steam will work just fine, for mycology, I'm not sure I want to cook TC agar that long, I'm looking at some of the hormones like TDZ.. and they will break down.


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## Voidling (Sep 29, 2015)

As far as hybrids evolving, I don't know how much further they can be taken. At least not until they start researching and selecting for different canababanoids. 

I had fallen for the seed company hype before and burnt out on their pitches of latest and greatest. If they would stabilize a line so that 90% of the seeds matched their description I'd be stoked. But one out of 50? Then the description is pointless.

I'd give a lot to have my old "blueberry" back. I'm not sure what it really was but smelled like lemon head candy, tasted similar and had a nice high. I typically hate the taste of any marijuana but that one wasn't so bad.


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## GrowerGoneWild (Sep 29, 2015)

I'm no genetics expert, but looking at all the types of beans out there with only around 22 chromosomes, and cannabis with only 20 I'm sure that there are plenty of undiscovered phenotypes. Keep in mind the drug type cultivar has been only been intensely bred within the last 100 years... 

I more or less have a fuzzy (I might be generous in saying that) idea of how to eliminate population variation with inbreeding to have a consistent population with homozygous traits. .. I would say that would be on the breeder for not releasing a inbred line with limited phenotype variation. Correct me if I am wrong but a IBL F1 would be more desirable? 

Kinda why I like clones. Especially with a production garden.

I'll have to say the lemon/citrus/grapefruit note is cannabis is almost too common. (Just my opinon) Not saying its bad, but the other fruit type aromas like pineapple, or blueberry.. seem very desirable. I for one lost a C99 that had that fresh pineapple aroma when pheno fishing. I kinda wish I had her back.


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## Voidling (Sep 29, 2015)

Oh yes, I had some hash sent to me of pineapple c99. Unfortunately two partners had a bad falling out and they both left the forum before I got seeds for some of their stuff. I am interested in the chocolate claims. 

Oh there will be variations in this and that but relatively small differences. I suppose looking at it as a connesuer then yeah ever slightly changing is there thing. But if one is just liking to feel better then just need something that works for you. I'm in the second group. I'm in extreme physical pain right now. 

An ibl is definitely on the breeder, but none of them go that far. Personally I'd rather not have feminized seed in case I want to cross my own


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## Voidling (Sep 29, 2015)

Just checked my stash. Got a couple c99 seeds. And a couple labeled c99 phenotype. Not sure if that's ibl or just off of a pineapple pheno mother.


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## GrowerGoneWild (Oct 20, 2015)

Moving forward finally on this. First formula will be from Melinda Davis.

Stage 1 Division/Stage 2 divisions

M&S medium with vitamins.
2 Tablespoons of sugar.
1ml PPM
.5ml IBA at 1mg/ml
1ml of Kintien.
9g/l Agar
30 g/l sucrose. 

This is very confusing how this is written in the book, so lets simplify.

Formula for 1 liter of Stage 1 & 2 divisions.

9 grams of MS medium with vitamins.
30 grams of sucrose.
.5 gram of IBA (Auxin)
1 ml of PPM (Plant Preservative Mixture) Biocides to keep the medium from getting contaminated
1 ml of Kiniten (Cytokinin)

Optional 1 drop of green food coloring, just for reference purposes.

Now unlike the chinese version done on commercial hemp, this has PPM in it.. An advantage for us that don't have a laminar flow hood. This should help reduce the contamination of the cultures. Im still going to PC the mixture and make a makeshift laminar hood.


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## Grandpa GreenJeans (Oct 21, 2015)

GrowerGoneWild said:


> Heh, yeah you beat me to it.. you dont need a PC, I've found 90 min of steam will work just fine, for mycology, I'm not sure I want to cook TC agar that long, I'm looking at some of the hormones like TDZ.. and they will break down.


At 15psi... u need the pc dude


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## Grandpa GreenJeans (Oct 21, 2015)

All my studies had stated "sterile".


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## GrowerGoneWild (Oct 21, 2015)

Grandpa GreenJeans said:


> At 15psi... u need the pc dude


PC for mycology or TC..? 

I'll be sterilizing the TC.. But I'll have to play with the sterilizing times, since PPM will be used in the media.


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## Grandpa GreenJeans (Oct 21, 2015)

GrowerGoneWild said:


> PC for mycology or TC..?
> 
> I'll be sterilizing the TC.. But I'll have to play with the sterilizing times, since PPM will be used in the media.


Correct. Both, myc and TC.
And also correct on playing with time and not temp. Psi must remain at 15 and no less than 220F


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## Grandpa GreenJeans (Oct 21, 2015)

Or stage serialization. 2 or 3 sessions
Similar to pasturization but at specs previously stated


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## Voidling (Oct 21, 2015)

I look forward to your experience. Wish I could get into it right now myself.

Did those sources I sent do you any good?


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## GrowerGoneWild (Oct 21, 2015)

Grandpa GreenJeans said:


> Correct. Both, myc and TC.
> And also correct on playing with time and not temp. Psi must remain at 15 and no less than 220F


For Myc, I've never PC cakes I've simply steam sterilized at 90 min at full boil. I think I've only had 2 possible contams. Out of the hundreds cakes I've run over the years. No starts is a bigger problem. I think its overkill but this is my data point. 

I already said I was going to PC the TC agar, The one formula has Plant Preservative Mixture, so this should give me more wiggle room with shorter sterilize times. My concern is denaturing the plant hormones, I might try a control group with no heat other than the bare minimum. We will see. 

The real weak point of TC is how the material is prepped prior to culture, in my opinion. Chinese standard says to wash with APSA80 (Amway soap) Melinda uses dilute bleach.. I'll have to come up with something economical, and that makes sense. I have a bunch of Chlorhexidine Gluconate soap, I might try that too.


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## Voidling (Oct 21, 2015)

Isn't diluted bleach economical? My understanding is that college labs teaching tc use flowers and use diluted bleach as well


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## GrowerGoneWild (Oct 21, 2015)

Voidling said:


> Isn't diluted bleach economical? My understanding is that college labs teaching tc use flowers and use diluted bleach as well


Im not sure if its going to damage the tissue.. seems like other techs are using other methods. But yes, bleach should be the cheapest way.. not sure if its the best way.


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## Voidling (Oct 21, 2015)

Yeah I'm sure it's a fine line of diluted enough to not hurt plant tissue but strong enough to kill pathogens.

I need to get some stuff to try myc. I spent two weeks going to a psychiatrist for ketamine treatment, way too expensive. Want to see if mushrooms well help with micro dosing.


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## Grandpa GreenJeans (Oct 22, 2015)

GrowerGoneWild said:


> For Myc, I've never PC cakes I've simply steam sterilized at 90 min at full boil. I think I've only had 2 possible contams. Out of the hundreds cakes I've run over the years. No starts is a bigger problem. I think its overkill but this is my data point.
> 
> I already said I was going to PC the TC agar, The one formula has Plant Preservative Mixture, so this should give me more wiggle room with shorter sterilize times. My concern is denaturing the plant hormones, I might try a control group with no heat other than the bare minimum. We will see.
> 
> The real weak point of TC is how the material is prepped prior to culture, in my opinion. Chinese standard says to wash with APSA80 (Amway soap) Melinda uses dilute bleach.. I'll have to come up with something economical, and that makes sense. I have a bunch of Chlorhexidine Gluconate soap, I might try that too.


You can always order the agar media and some come with anibiotics in the mix to prevent or prolong micobial growth while you administer the growth hormones along the way. As I'm sure you already know, there's multiple hormones needed for a TC, and each has a specific function. Grow roots, grow stems, and leaves. Orchid enthusiasts and forums alike have a good deal of info on TC and give reputable retailers for the equipment you'll need.


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## GrowerGoneWild (Oct 22, 2015)

Grandpa GreenJeans said:


> You can always order the agar media and some come with anibiotics in the mix to prevent or prolong micobial growth while you administer the growth hormones along the way. As I'm sure you already know, there's multiple hormones needed for a TC, and each has a specific function. Grow roots, grow stems, and leaves. Orchid enthusiasts and forums alike have a good deal of info on TC and give reputable retailers for the equipment you'll need.


PPM (Plant Preservative Mix) is the biocide I will be using for the TC I dont know if it also has antifungal qualities. I'm going with a generic MS medium with vitamins. 

The cytokinin's and auxins will be mixed into the agar.. and will be done in stages. Rooting agar will be a different formulation, and will be marked with a different color.


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## Grandpa GreenJeans (Oct 22, 2015)

What type of containment vessels are you using?


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## Grandpa GreenJeans (Oct 22, 2015)

Where will your samples come from for the culture? Roots, phloem, cadmium, zylem


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## GrowerGoneWild (Oct 22, 2015)

Grandpa GreenJeans said:


> What type of containment vessels are you using?


I got lots of 1/2 pint jars, but I'd like to use Polypropylene containers (PP) cheap and plentiful, and they can be sterilized. and the top is transparent, I figgure I could get about 5 to 10 explants into the container.


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## Keighan (Oct 22, 2015)

Uncle Ben said:


> You have a $200,000 lab complete with centrifuge, etc?


Just saying dont care the reprecussion from this you are a very intelligent grower and alot have learned alot from you but youre seriously one of the few sore thumbs in this stoner idiolistic society we like to think of it as going against the grain and being a conceited douche frankly, ive seen you do it on tons of threads to people who didnt deserve it and werent talking about or to you, cant everyone just be peaceful, informitive, and helpful?...really


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## Grandpa GreenJeans (Oct 22, 2015)

Keighan said:


> Just saying dont care the reprecussion from this you are a very intelligent grower and alot have learned alot from you but youre seriously one of the few sore thumbs in this stoner idiolistic society we like to think of it as going against the grain and being a conceited douche frankly, ive seen you do it on tons of threads to people who didnt deserve it and werent talking about or to you, cant everyone just be peaceful, informitive, and helpful?...really


You forgot supportive. Lol just sayin.


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## Keighan (Oct 22, 2015)

Grandpa GreenJeans said:


> You forgot supportive. Lol just sayin.


My bad I was trying to explain my frustration without appearing as rude. Or atleaste as respectful as I feal I could be.


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## Grandpa GreenJeans (Oct 22, 2015)

Keighan said:


> My bad I was trying to explain my frustration without appearing as rude. Or atleaste as respectful as I feal I could be.


I wasn't put off by it. You make a very valid point, that were all here to share teach, help and support each other. Cannabis has a weird way of bringing people together and we all share the same passion for the plant. Sometimes it's good to remind people politely were all here for the same reason. Even though old timers (like myself included) have been known to have inflated egos, we're all the same.


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## Keighan (Oct 22, 2015)

And the new generation like me is beyond grateful for all of your "old timers" knowledge, Im constantly throwing my pride in the trash and asking questions or asking the local guerilla growers. Its a beautiful experience..new age og.


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## Grandpa GreenJeans (Oct 22, 2015)

GrowerGoneWild said:


> I got lots of 1/2 pint jars, but I'd like to use Polypropylene containers (PP) cheap and plentiful, and they can be sterilized. and the top is transparent, I figgure I could get about 5 to 10 explants into the container.


Mushbox.com has the containers I believe. I've bought from him 5 years ago and still have the PP jars. If your into mycology and isolate your mycelium on petri, I have had good luck with his nutrient agar. Pre made and sterilized.


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## GrowerGoneWild (Oct 22, 2015)

Keighan said:


> Just saying dont care the reprecussion from this you are a very intelligent grower and alot have learned alot from you but youre seriously one of the few sore thumbs in this stoner idiolistic society we like to think of it as going against the grain and being a conceited douche frankly, ive seen you do it on tons of threads to people who didnt deserve it and werent talking about or to you, cant everyone just be peaceful, informitive, and helpful?...really


UB's comment is so far removed from reality.. 200K to make cannabis tissue clones?. centrifuges?.. 

He does have some knowledge as a gardner, but when he says stuff like that it makes him sound like a keyboard warrior. 

Anyways, we are all just gardeners, and farmers.. with different viewpoints, so no real reason for anybody to get a chip on their shoulder. But then again this is "troll it up".. More effort towards dissrespect than discussion.

Anways back to Tissue Culture


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## GrowerGoneWild (Oct 28, 2015)

Grandpa GreenJeans said:


> Where will your samples come from for the culture? Roots, phloem, cadmium, zylem


I'm going to start off with some fan leaves, cut that up into its natural sections, and use small shoots from the main stem that would be deemed too small for clones.


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## Grandpa GreenJeans (Oct 28, 2015)

GrowerGoneWild said:


> I'm going to start off with some fan leaves, cut that up into its natural sections, and use small shoots from the main stem that would be deemed too small for clones.


That sounds like a good start. You can virtually take the tissue from anywhere on the plant for a TC. I have seen alot of orchid cultivators take the inner most tissue. They target new growth, i guess its because it has the highest concentration of growth hormones.


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## coconutbeach (Jan 3, 2016)

Grandpa GreenJeans said:


> Mushbox.com has the containers I believe. I've bought from him 5 years ago and still have the PP jars. If your into mycology and isolate your mycelium on petri, I have had good luck with his nutrient agar. Pre made and sterilized.


Four and six ounce polypropylene and glass jars are best for gel culture. We are also using 23mm plastic tubes for introductions and starts. All are included in the Microclone tissue culture kits. planttc.com


Grandpa GreenJeans said:


> That sounds like a good start. You can virtually take the tissue from anywhere on the plant for a TC. I have seen alot of orchid cultivators take the inner most tissue. They target new growth, i guess its because it has the highest concentration of growth hormones.


Make it easy and use 1-1/2" clean vegetative branch tips.


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## coconutbeach (Jan 3, 2016)

GrowerGoneWild said:


> UB's comment is so far removed from reality.. 200K to make cannabis tissue clones?. centrifuges?..
> 
> He does have some knowledge as a gardner, but when he says stuff like that it makes him sound like a keyboard warrior.
> 
> ...


Startup cost of Microclone tissue culture. Kit, pressure cooker, tub, rack lights. Less than $400 for three months growth and first 150 rooted clones.


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## coconutbeach (Jan 3, 2016)

GrowerGoneWild said:


> Meh, I've run sugarless mediums like in the case of psylosybin cultivation, you can have contamination take hold if the medium has sugar in it or not. It might decrease the likelyhood of infection, so point taken.
> 
> Actually the method is not like mushroom cultivation, the byproduct of fungi is CO2, and it does not take much airflow, in fact on the Brown Rice Flour (BRF) cakes I make I do not expose them to circulating air until a fruiting body shows.
> 
> ...


Check out Microclone tissue culture kits at planttc.com or at Alaska Jacks in Palmer.


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## coconutbeach (Jan 3, 2016)

Voidling said:


> Isn't diluted bleach economical? My understanding is that college labs teaching tc use flowers and use diluted bleach as well


A typical wash is 10% bleach for 15-20 minutes, however, we much prefer 500ppm of dichlor because the Cl evaporates and typically does need the additional sterile water soak. Dichlor is included in the Microclone TC kits.


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## coconutbeach (Jan 3, 2016)

thump easy said:


> im gona try it at our new place im gona invest in all the steril equipment and just for shits and gigles im gona try to market them lolz who knows it might work as far as genetics go sounds like another project new place almost doneView attachment 3336537View attachment 3336538 View attachment 3336541


What a great space. I am in CO as much as I am at home these days. Your space can make all the clones you need and you will likely still have space left. You will need a filtered air area for growing and handling, kitchen for media prep and dishes, and rooting trays and racks. OUr biggest setups still started with a Microclone kit and scaled up from there. See how you can HEPA filter the ducted air at one end and curtain it off for TC racks. 

These are the racks we made to fit the width of the four foot shelves. We call them Microclone Rack Trays.


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## coconutbeach (Jan 3, 2016)

AlphaPhase said:


> it's basically for preserving genetics. If you want 1000 genetics in a small warehouse, tissue culture is where it's at. It works great. I haven't done this myself, but I know people who have. I wouldn't take a tissue culture for something you want to flower next month though, but it does have it's use.


Check out Microclone Cannabis Tissue Culture. Search Google.


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## coconutbeach (Jan 3, 2016)

eyes13 said:


> Ideally I want an incubator in addition to the same veg space I use now, so just eliminating a dedicated mother space for ~20 strains I'd wish to hold on to. Does that sound like a reasonable application here? Glove box, filter etc are within reach.


The first Microclone grow racks held multiple vessels of over 30 varieties on one 2x4 ft shelf under two 54-watt t5's.


Sativied said:


> Nice job thump! I look forward to more info.
> 
> I've seen attempts and journals at several forums and they typically end before it becomes interesting.
> 
> ...


Regular clone ROOTING is much faster because of the resources put into creating the 6" + cuttings. We still always have some rooted clones to back up the cultures and prove the vigor. It is all of the growth, potential, and advantages that tissue culture presents that should be explored and utilized. Tissue culture is basically growing plants with sugar. The rest of the items we love are there too like minerals and vitamins. Using sugar means we don't need roots to grow plants and create new cuttings but that we will also need to prepare clean media, clean plants before introducing into culture and handle with clean tools in a clean area. 

Before I scare anyone by using clean so much, I regularly do tissue culture on the road in hotels, countertops, and on folding table demonstrations at hydro and cannabis trades shows. Tissue culture is how the commercial horticulture industry clones nearly all of their plants and the advantages are ideally fitted to cannabis. Hundreds of clones are produced from only 8 sf under 100 watts of light every month. And we typically stack those racks five or six high for the big hitters. One outfit has even added another three feet to their wire baker's racks to go to nine feet and eight shelves per rack.


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## AlphaPhase (Jan 3, 2016)

coconutbeach said:


> Check out Microclone Cannabis Tissue Culture. Search Google.


Sure will. Thanks for the tip. I plan on incorporating this method on my next move to a place with more space. I know it'll take a while to get the hang of it, but sometimes I just want to put a clone on the back burner for a while, but keep it, but keeping a mama plant I don't plan on flowering for a while really takes up time and space. Tissue culture ftw


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## coconutbeach (Jan 3, 2016)

mudballs said:


> i reviewed that and all that paper describes is the most expensive method to root clones.
> native humbolt has the most pertinent info to proceed. sterilization and proper vitamins mix.
> i'm gonna send you to this thread
> https://www.rollitup.org/t/scarholes-root-pruning-sex-reversal-method-for-fem-seeds.853829/
> in this thread scarhole discusses a prominent breeder culturing root cuttings. if you go down that rabbit hole you might find a way of just taking a cutting from a root and growing a plant.imagine how many more clones you could get from a mother? i'm fascinated by it but not in a position to pursue it at the moment.


Your wish is granted.


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## coconutbeach (Jan 3, 2016)

MikeGanja said:


> Mudballs, I agree. It looks like the process probably could be made at home to a somewhat reasonable cost but the kits like the one in the link doesn't guarantee a successful clone with cannabis. I would be more convinced if Bill Graham show us examples with cannabis instead of redwood.
> 
> Native Humboldt, great news that I could get the needed equipment for $ 100. It's less then half the price I found so far.. Sorry to hear that you didn't succeed in finding a working formula for cannabis.
> 
> Some Asian scientists have published a study on tissue cloning of cannabis. Do you think it can be useful for growers like us? --> http://www.pakbs.org/pjbot/PDFs/41(2)/PJB41(2)603.pdf


Hi Native. It would be my pleasure. The instructions are the same and we will be producing a cannabis tissue culture kit as soon as we get the legal "go". Here are some TC cannabis pics of the kits in action and a tip sheet for making starts in liquid medium. The media in the Microclone kit are excellent for cannabis as well as enough other plants and is still the most popular cannabis TC media made. Thank you and good luck.


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## Sativied (Jan 5, 2016)

coconutbeach said:


> Regular clone ROOTING is much faster because of the resources put into creating the 6" + cuttings.


Yes. Which makes TC attractive if putting resources into those cuttings would cost so much in resources (space and light for mother plants) that it justifies TC, which only makes sense when you need shit loads of clones, but then, if you need those for yourself it means you veg and flower a shitload of them too, which gives you plenty of branches (need only 1 per plant in theory, a few for backup) to use as cutting, especially when the plants are subject to some canopy control such as topping. 



coconutbeach said:


> Hundreds of clones are produced from only 8 sf under 100 watts of light every month.


Hundreds of plantlets. The required space and light for any amount of "clones" depends on the size of those clones and not on whether they are from tc plantlets or regular cuttings. 

I totally get the appeal and TC has its useful purposes but in practice TC requires additional space and resources, besides time. 

The increase in numbers is from the ability to make many from little, as in many plants from a single leaf or a calyx for example. It's not about being able to produce the same number of clones in less space. In practice it's particularly useful for companies who sell millions of clones to thousands of growers.

Growing a cutting in sugar an then calling it tissue culture, ending up with a weak clone, is almost as silly as some other guys at another forum pretending to do tissue culture with seeds in agar...


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## GrowerGoneWild (Jan 5, 2016)

^^^^ 

You're right, TC does not yield a superior cutting, if you observe the premium cuts offered by Dark Heart Nursery vs the TC clones, they have thinner stalks vs the conventional cuttings. They are using TC for the high volumes needed for a commercial cloning operation.

If cannabis was an unregulated plant, it would make more sense to simply take clones in a conventional manner. Since no specialized equipment is needed. TC is more or less for plants that are hard to duplicate, as observed in the agri industry. 

Cannabis hybrids could still benefit from TC: 

-Pathogen free stock.. 
-Duplication hard to clone cannabis strains.. like Ruderalis hybrids.

The seed to agar method could explain that particular method of TC. 

Another thing TC could be used for is dealing with "duds" or old cuttings, or virus infected stock. The theory is that newest growth is less likely to be infected with pathogens, TC could be use to create clean clones. And people seem to be talking about TC giving vigor to dudded out cuttings.


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## Sativied (Jan 5, 2016)

You had me at "you're right" already and then you put "duds" between quotes too... but then you mentioned cloning ruderalis... Ah well good post anyway.

And yes, rejuvenation of cuttings stripping the results of (mis)nurture from nature could be one of the more useful purposes of tc for a tiny amount of growers. Not enough to make it common practice for cannabis growers though.

Many of the large commercial cannabis grow ops today are just inflated homegrows. Once pros take over the industry TC for cannabis may become more common but I don't think it will ever become common for home and other small growers. Those [email protected] kits are a bit silly unless it's for educational purposes. Practical it is not.


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## GrowerGoneWild (Jan 5, 2016)

Sativied said:


> You had me at "you're right" already and then you put "duds" between quotes too... but then you mentioned cloning ruderalis... Ah well good post anyway.
> 
> And yes, rejuvenation of cuttings stripping the results of (mis)nurture from nature could be one of the more useful purposes of tc for a tiny amount of growers. Not enough to make it common practice for cannabis growers though.
> 
> Many of the large commercial cannabis grow ops today are just inflated homegrows. Once pros take over the industry TC for cannabis may become more common but I don't think it will ever become common for home and other small growers. Those [email protected] kits are a bit silly unless it's for educational purposes. Practical it is not.


I'm not knocking on the homegrower, because that includes myself. But its nice to to have that tool if needed.. I would rather mix my own agar for educational purposes, and to see if its something that could be used on a commercial scale. 

Anybody should be able to observe in industry that some operations are specialized.. same applies to agriculture, TC is one of those, thats pretty much all they do, if a farmer/gardner needs some plants they simply go to the nursery. Seems like misuse of resources for a warehouse grow to propagate cuttings with the TC method when they should be using that for flowering.


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## GrowerGoneWild (Jan 14, 2016)

Just a couple of notes I'd like to share about tissue culture when working with hormones.

1) The ammounts in TC are very small, you'll have to be careful with your math and you'll need a accurate scale to weigh the small ammounts.

2) IBA does not dissolve in water.. It will dissolve in 100 proof ethanol (I like smirinoff). Then you can dilute it with water to mix into your agar.

3) Kinetin does not dissolve in water, you will need to mix a caustic solution to dissolve it. 1 teaspoon of Sodium Hydroxide (Lye) will work to 1/4 cup of water, dissolve the lye first, then add the kinetin. Then dilute with water. General Hydroponics PH UP will not work. 

Be very careful with any type of strong caustic, when pouring the lye grains, when I poured it into a dish, some of them bounced out of the shallow pyrex dish, lye isn't active until you touch it or it gets wet then it will burn the crap out of you.


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## thump easy (Jan 17, 2016)

When you guys figure it out let me know?? Its easy but it tskes to long in my opionion..


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## greenghost420 (Jan 17, 2016)

too long for me homie, i ditched that shit lol ill just keep cuts in the fridge if i need to...


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## GrowerGoneWild (Jan 17, 2016)

thump easy said:


> When you guys figure it out let me know?? Its easy but it tskes to long in my opionion..


I got it figgured out, if you ever get around to posting your formula, i'm all ears.


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## thump easy (Jan 17, 2016)

Ill try but it might be a while


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