# Plant tissue culture as a way to clone on a mass scale??



## K1Ng5p4d3 (May 15, 2009)

Hey guys, 

Ive been reading a bit about the whole Plant tissue culture phenomenon, and i was wondering if this method would work very well with cannabis. There are more expensive kits out there, but this is the only one i could find that has a cut n dry description of how it works, for the people out there who havent heard of it yet.

http://cgi.ebay.com/Plant-Tissue-Culture-Cloning-Budget-Starter-Kit_W0QQcmdZViewItemQQitemZ220413524721QQsalenotsupported

Im just curious if it would be effective for somebody who's looking to take like 500 clones at once, without having to have the 20 mother plants that would have to come with it in normal circumstances. If i read everything correctly, i wouldnt even really need ONE mother. So, any opinions on this would really help me out, because it would make things worlds more efficient and convenient for me if this were a plauseable method to apply to a SOG setup. 

Space is going to get cramped in my growroom, and not having to have a mother tent would really make things WAY easier on me. But please give your honest opinions only, i dont wanna scrap the mothers i already have going, nor will i, until i know that this method works, and works well. 

Thanks for your feedback, 
K1.


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## mrshark (May 16, 2009)

i am considering trying this myself


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## mrshark (May 16, 2009)

a moderate shelf space in a clean room and modest lighting and you could culture 500 plants easily. however you must and i stress must be Anal about cleanliness. anything gets into the jar and you can scrap it
everything must be clean. now if i can just find the proper mix of agar and nutes to make it fly


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## KP2 (May 16, 2009)

mrshark said:


> a moderate shelf space in a clean room and modest lighting and you could culture 500 plants easily. however you must and i stress must be Anal about cleanliness. anything gets into the jar and you can scrap it
> everything must be clean. now if i can just find the proper mix of agar and nutes to make it fly


bs. clean isn't the word. you must be STERILE! if you don't have a chem lab in your house, don't even think about it.

i love how micro culture comes up around here each month. everyone rediscovering that gravity does in fact work...


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## K1Ng5p4d3 (May 16, 2009)

you haved to be sterile, yes, but you dont need a chem lab. If you'd check out the newer kits that are around today, you'd see that alot of them are boasting that very same fact dude. N i never said that i just discovered it brother, ive known about it for a while, i just never considered using it until i had the thought that i could replace the need for mother plants, by using this method, which is totally plauseable, it would just take a little longer for my clones to get up off the ground. If i were to work it into my perpetual schedule though, and push everything back by a month, i'd still be able to go an lb/two weeks if i wanted to. I just wanted to know if there were people out there who agreed with me on the matter.

There's a synic everywhere ya go nowadays, lol....


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## lolapug2175 (May 16, 2009)

keep in mind that tissue propagation can/does take more than 2/4 months for usable starts to develop. It is a myth that mothers can be negated. raw genetic material must be obtained freshly batch to batch. alternatively all that is required can be obtained from a few leaves. Honestly from your description of needs.. 500 just does not warrant this process. Also sterility cannot be over emphasized! tooling the process is expensive unless you have sizable autoclave laying around. the vendors try to make the kits look easy... However, it takes approximately 4-6 months to achieve consistent deliverable results.
Good Luck!


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## TMB77 (May 16, 2009)

Hey King

I currently work in plant tissue culture/propagation/transformation. I am intimately familiar with all the necessary steps and components, and can pretty much answer any questions you'll have on this subject.

The link you provided gives no details about what they're actually selling you. And trust me, 59.95 aint gonna get you SHIT.
Things you would need to do this correctly:
-A pressure cooker to sterilize containers
-A sterilizable space (I use a laminar flow hood) but you could possibly create a space where the ambient airborn contaminants wont get in the agar. The thing is, you'll not know if your area is sterile until you see whether fungus is growing on the agar, and if it is that will ruin the sterility of that plant and it will have to be tossed.
***Something people dont realize: You need to START with sterile plant matter. WHich means you have to sterilize a seed in 1:2 clorox, sterile water, and a few drops of one of a few types of scientific detergent (at least I assume it's 'scientific' because i've never seen "Tween" anywhere but in the lab). You have to grow the seed sterily, and take cuttings from that. 

you'll also need:

-Plant hormones (auxins, cytokinins)
-A gelling agent (agar, gelzan, gelrite, there are a few you can use)
-MS vitamins w/micronutrients
(the above things are the absolute base needed to make a nutrient rich growing medium with the necessary components)
-A magnetic stir plate
-Assorted labware (glass beaker, a few ehrlenmeyer flasks, etc. The hormones have to be added AFTER the steam sterilization of the media, after it's cooled to 50 degrees, so that can be tricky at home.
-Tools that can be sterilized
-enough vessels to do all the cloning you have planned.


Also, there is something called "somaclonal variation". which can/does occur to plant material that is kept in tissue culture for too long. http://en.wikipedia.org/wiki/Somaclonal_variation so keeping a mother in tissue culture too long could start changing your product. (and probably for the worse)

I dont really get what your plan is, you cant keep a mother in those vessels too long (because of what I already mentioned) but also because it is going to keep growing, and VERY quickly outgrow any sort of small vessel you're using, so you'd have to be subculturing (taking some leaf material, putting it in a new jar and growing roots and shoots from it) which would lead to a lot more somaclonal variation. Also cuttings would take a while to form the shoots and roots you want. You'd have to put it in one media formulation to produce shoots, then move it to another one to induce roots. It is probably much more complicated than you might have thought.

anyways, I gotta run. Hopefully i've helped educate without damping your spirit too much. This CAN be done, but it's more complicated than people tend to think.


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## smoke and coke (May 16, 2009)

K1Ng5p4d3 said:


> Hey guys,
> 
> Ive been reading a bit about the whole Plant tissue culture phenomenon, and i was wondering if this method would work very well with cannabis. There are more expensive kits out there, but this is the only one i could find that has a cut n dry description of how it works, for the people out there who havent heard of it yet.
> 
> ...


that is a starter kit. after 30 days you will use the super kit and its around $253.00 for that. 
http://www.planttc.com/


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## SINGLECOLA (May 19, 2009)

I do PTC in school and at home. Go to http://www.kitchenculturekit.com/ and you can use the free how to using household stuff. Also has links you can go to and buy the couple hormones and plant preservatives you will need, they are super cheap. I get mine from school. 
How about 100's of exact replicas in 2 months. ready to plant into what ever medium you want. Start searching craigslist for baby food jars, your going to need hundreds. PTC is very addictive! Think of it like this; you are forcing every cell that makes up that little piece of pot leaf, to grow into a plant. Thousands of cells make up a little 1/4 x 1/2 inch piece of leaf, so find jars as fast as you can and get ready!


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## SINGLECOLA (May 19, 2009)

p.s. Everyone's a critic and yes you will have a contaminated one or two but for the most part if your slightly competent and can read and follow direction you will have great success. Before I did Plant Tissue culture, I was told the same thing about home mycology and right now I am still growing mushrooms in a home "sterile" environment using Rubbermaid bins as terrariums and one placed on its side as my clean room while I work. Its the same bin I use when I do PTC and I rarely have problems.
http://www.kitchenculturekit.com/
Its what I used in my introductory Horticulture 101 class and had no problems. 
Just enjoy what your doing and stay positive.


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## TMB77 (May 19, 2009)

SINGLECOLA said:


> p.s. Everyone's a critic and yes you will have a contaminated one or two but for the most part if your slightly competent and can read and follow direction you will have great success. Before I did Plant Tissue culture, I was told the same thing about home mycology and right now I am still growing mushrooms in a home "sterile" environment using Rubbermaid bins as terrariums and one placed on its side as my clean room while I work. Its the same bin I use when I do PTC and I rarely have problems.
> http://www.kitchenculturekit.com/
> Its what I used in my introductory Horticulture 101 class and had no problems.
> Just enjoy what your doing and stay positive.



Nice, looks like they put together a pretty decent package there. It's good to hear someone's doing it at home without a bunch of headaches. 

I'm surprised you can successfully sterilize a vessel and medium in the microwave, haha, i'm gonna have to give that a try.

Singlecola, do you use the microwave for sterilization? What about your instruments?
How do you sterilize the hormones...they wont stand up to an autoclaving, so they have to be filter sterilized and added after the media has cooled. Thats so sweet if a microwave will sterilize the media without deactivating the hormones.


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## dthed (May 19, 2009)

KP2 said:


> bs. clean isn't the word. you must be STERILE! if you don't have a chem lab in your house, don't even think about it.
> 
> i love how micro culture comes up around here each month. everyone rediscovering that gravity does in fact work...


 No need to be nasty and cocky up in here! Save it for somewhere else. BTW, What does a chem lab have to do with it? No need to lead people on thinking they need a "chem lab". Shure you should be sterile but 'clean' is ok to use as a word too.


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## Uncle Ben (May 21, 2009)

K1Ng5p4d3 said:


> Hey guys,
> 
> Ive been reading a bit about the whole Plant tissue culture phenomenon, and i was wondering if this method would work very well with cannabis. There are more expensive kits out there, but this is the only one i could find that has a cut n dry description of how it works, for the people out there who havent heard of it yet.
> 
> ...


I've asked Dutch seed vendors why they don't use meristem/mericlone labs to clone their best, and it seems no one is interested. Seeds carry a higher value, are easier to ship, etc. I'm talking even 12 years ago when cannabis culture started to ramp up on the internet. Being an orchid grower who would buy mostly mericlones that carried the highest awards by the American Orchid Society (AOS), I knew the process well.

There are quite a few labs in the States that will clone using meristem tissue for a fee, but with cannabis, you're mileage will vary. 

Good luck,
UB


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## canndo (Mar 12, 2011)

I wonder why they are not interested in doing it for their own cloning needs.


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## Uncle Ben (Mar 13, 2011)

canndo said:


> I wonder why they are not interested in doing it for their own cloning needs.


Cost and time. For starts, you do need a completely sterile setup and they aren't cheap. You need a special centrifuge, agar, lab materials and equipment, etc. When you can bust someone's bank account regarding 10 seeds taken from a mama mutt that produced 200 seeds at practically no cost, why bother?


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## canndo (Mar 14, 2011)

Ben, My serile room cost less than a thousand dollars, my hood about 500. I don't use a centrifuge, the agar is about 25 dollars. While I can see your point about simply selling seeds, I can't see them sitting around snipping tips and jaming them in rock wool over and over and over and over and over and over again.


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## Uncle Ben (Mar 14, 2011)

canndo said:


> Ben, My serile room cost less than a thousand dollars, my hood about 500. I don't use a centrifuge, the agar is about 25 dollars. While I can see your point about simply selling seeds, I can't see them sitting around snipping tips and jaming them in rock wool over and over and over and over and over and over again.


I understand. Trust me, this is old potatoes for me. Breeders/cross dressors just aren't interested and I can see why - producing seeds is the only way to go when it comes to profit, and that's all this biz is about.


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## canndo (Mar 14, 2011)

Don't get me wrong Ben, I believe you, it just seems that they would save money if they micropropagated internaly.


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## Uncle Ben (Mar 15, 2011)

This fella explained it best. You must use fresh, healthy meristem material and the procedure must be done under sterile lab conditions. Ever seen pictures of a typical Dam grow room lol? 



lolapug2175 said:


> keep in mind that tissue propagation can/does take more than 2/4 months for usable starts to develop. It is a myth that mothers can be negated. raw genetic material must be obtained freshly batch to batch. alternatively all that is required can be obtained from a few leaves. Honestly from your description of needs.. 500 just does not warrant this process. Also sterility cannot be over emphasized! tooling the process is expensive unless you have sizable autoclave laying around. the vendors try to make the kits look easy... However, it takes approximately 4-6 months to achieve consistent deliverable results.
> Good Luck!


It's all about profits. One must eat their plant losses, address shipping issues, worry about competition from seedbanks, etc. There's enough bullshit a seed vendor has to put with because some noob doesn't understand how to properly grow a plant from seed and then expects a refund. Can you imagine how much grief (and bad press) a firm would get considering the losses from fresh plant material that was shipped for hundreds, perhaps thousands of miles under all kinds of weather conditions? One freeze experience in a UPS truck and they're toast. You can ship seeds under the most adverse conditions and still expect them to be viable upon arrival.

UB


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## canndo (Mar 15, 2011)

Ben, so far as my experiences are concerned, shipping plantlets rooted in media is asking for failure - hardening off in vitro plants is not for the faint of heart and I can't see very many people popping their newly purchased purple kushie kush OG into a plug and taking it from there. 

Your quote however, is a little misinformed from my perspective. Firstly, leaves make poor propagation material in that leaves require one to produce callus and then from there shoot and finally root. I have not been able to figure out the combinations of auxins and cytokinins to bring callus back to shoots. I am reminded of the land before Oss and Orick published a little book on how to grow mushrooms - before then, it was just too monumental a task. They managed to reduce the process to something everyone could do in their kitchen. Sterile conditions seemed insurmountable but they are not. I have never used an authentic autoclave and my pressure cooker works just fine. 

Beyond that, there are chemicals available that limit contamination in the medium to the point where I believe no glove box or hood is even necessary. As far as the need for fresh material? Well, I havn't needed such yet but we await final results. 

Note what I said though, I would think that a large facilty might use these techniques internaly. I have never believed that tissue propagation would replace seeds and I don't think that anyone would believe such a thing. I am beginning a new experiment (it is nice that this hobby takes so little time, space and money in that I can run multiple experiments simultaniously). I am going to take seeds and grow entirely in vitro from seedling to rooted plantlet. If I can do this, I suspect that I can clone dozens of plants before traditional methods can even yield a single cutting. The trick here is the ability to sex plantlets - I intend to start that this week. Thanks for the ongoing discussion Ben. I would be curious to know what you think of my personal efforts in this realm.


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## Uncle Ben (Mar 15, 2011)

canndo said:


> Ben, so far as my experiences are concerned, shipping plantlets rooted in media is asking for failure - hardening off in vitro plants is not for the faint of heart and I can't see very many people popping their newly purchased purple kushie kush OG into a plug and taking it from there.
> 
> Your quote however, is a little misinformed from my perspective. Firstly, leaves make poor propagation material in that leaves require one to produce callus and then from there shoot and finally root. I have not been able to figure out the combinations of auxins and cytokinins to bring callus back to shoots. I am reminded of the land before Oss and Orick published a little book on how to grow mushrooms - before then, it was just too monumental a task. They managed to reduce the process to something everyone could do in their kitchen. Sterile conditions seemed insurmountable but they are not. I have never used an authentic autoclave and my pressure cooker works just fine.
> 
> ...


You're not describing the mericloning process I'm thinking about. Check out orchid mericloning and you'll see what I mean. I talking about taking the apical meristem material, putting it into agar, spinning it in a centrifuge to induce asexual cell reproduction, spinning it again so they divide again, repeating the process until you have hundreds of identical, tiny plantlets after which you carefully......


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## canndo (Mar 15, 2011)

Ben, I don't go to the single cell, that, in my case would be redundant. I needn't isolate the meristem alone primarily because I do not fear viral infection. One needn't go down to that level in order to get mothers and clones - micropropagation. I suppose I could but then your quote would be more accurate - it would take months to get enough biological material. Again, there is no need to do this on a cellular level as far as I can see. I am now in the 5th generation from cutting to explant to plantlet with no degeneration I can see other than what I've experienced with hyperhydration. It seemed to me that the fellow you were quoting fears sterile conditions more than is warranted. Bioreactors have their place but now we are talking about either collecting secondary metabolites which won't much work with this particular plant - or generating many more hundreds of thousands of clones than anyone might need for this sort of work. There is a process whereby pollen is grown into haploid plants or treated with chemicals in order to make them diploid that seems interesting but I can't see the point in that except to preserve the exact genetics of a single remaining male plant.


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## Wolverine97 (Mar 15, 2011)

I'd really like to see this discussion furthered, I'm intensely interested. 

I have a "golden child" that exists only in clone form, and I believe it may be infected with a mosaic virus. It grows fast enough that it never becomes an issue, but if I try to keep plants in a quasi suspended animation it becomes evident. I'd dearly like to clean the genetics if possible, and it'd be worth a modest investment to do so.


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## canndo (Mar 15, 2011)

Woverine, in that case meristemic tissue is your answer. As you have probably already noticed, the vascular system of the plant grows slightly faster than the virus can infect so the very tips of your growing shoots are virus free (probably). It is a dicey move but you can afford to fail as your plant is still alive. If you wish I can take you throgh the steps and see if it might be right for you.


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## Wolverine97 (Mar 15, 2011)

canndo said:


> Woverine, in that case meristemic tissue is your answer. As you have probably already noticed, the vascular system of the plant grows slightly faster than the virus can infect so the very tips of your growing shoots are virus free (probably). It is a dicey move but you can afford to fail as your plant is still alive. If you wish I can take you throgh the steps and see if it might be right for you.


Oh for sure, yeah, that's actually why I've been looking into tissue culture because it's always been described to me as using the apical meristem, to obtain the pure and clean genetics. Couldn't I just disinfect a very small, soft-tip cutting of the apical meristem, and root it as normal? I actually did this with my last round of cuttings, but then I didn't isolate that clone from the rest. The ones I took may have been too large though (2-3" overall), as I'm not sure what's the largest I could do and still ensure that it's clean.


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## canndo (Mar 15, 2011)

I really don't know how small the bit of meristem you could root conventionaly. One of the problems I had to resolve is exactly what size is the right size to do this. Too small and it takes too long to grow - it also is easier to just kill the tiny explant with all the harsh treatment involved in sterilization. We have a secret advantage over most micropropagators in that the vast majority of the plants we grow are in clean conditions from the start - we don't have to go outside in all that filth. You could further protect your meristem by putting a clean baggie over a prospective tip and let it grow in even more sterile conditions. Too large and you will include the virus in the plant and gain nothing. I am not sure what size would be best in your situation - you can see what size I have found to work best in my experience - and it is far larger than all the texts I have read suggest. I get my best results with 1.5 to 2 inch, juvinile as possible, lower stem cuttings with a bit of larger stem still attached for surface area. But you need something much smaller I suspect.


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## Uncle Ben (Mar 15, 2011)

canndo said:


> I really don't know how small the bit of meristem you could root conventionaly..


It's not an issue of rooting meristem tissue. Do some research. 

UB


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## canndo (Mar 15, 2011)

Ben, Wolverine asked "Couldn't I just disinfect a very small, soft-tip cutting of the apical meristem, and root it as normal? I actually did this with my last round of cuttings, but then I didn't isolate that clone from the rest". And I answered in the way I did.


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## ulis5714 (Mar 17, 2011)

Do leaves have to be fresh, or can leaves be frozen for use later?


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## canndo (Mar 17, 2011)

Ulis. I have not been able to do anything with leaves, they form callus but nothing else. I have found recent studies that indicate rooting from the leaf callus but shoots from that callus are if I recall, in the 1 percent range. But the other answer to your question is that if you do use a leaf to generate callus, it won't work if the leaf is frozen or dried - it has to be alive and the fresher the better.


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## ulis5714 (Mar 17, 2011)

OK. I was looking at a plant tissue culture kit from Discount-Hydro.com, but it could be beyond my capabilities. I best leave such things to the experts!


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## ulis5714 (Mar 17, 2011)

I'm a new member, and I thought I replied, but maybe I have not learned to use this site properly yet. So, I have been looking at a PTC kit online, and though it looks good, cloning is probably the way to go.


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## canndo (Mar 17, 2011)

Ulis, this all depends upon what you wish to do. Take some cuttings off of a few moms, then no, this may not be "beyond" you but there is no reason to go through the trouble and expense.


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## canndo (Mar 17, 2011)

One more thing, as I said, by all means the kit is a good thing and it will certainly help, but the media mix is not even close to being appropriate for our plant of interest.


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## ulis5714 (Mar 17, 2011)

It's good to be able to ask for good advice! I'm strictly a small-scale grower, everything I grow will be only for me, and PTC seems to be for commercial growers! Once upon a time I thought I could freeze a good plants leaves for later, but thanks for setting me straight!


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