Tissue Culture Cloning

[PigSquishy]

I have been learning more about a process called Tissue Culture Cloning, which to break it down and just tell you what it means is simply this... Imagine taking one leaf off your plant and cutting it up into 50-clones from just one leaf and growing an entire plant from just that little tiny piece of a leaf and be able to keep mother plants in stasis that are so tiny you could keep them in a shoebox, really to be grown into full sized plants when ready. While it all sounds really cool the reason more people aren't off doing it is because it takes some knowledge and very sterile working conditions. So I've been doing my homework, and digging into the process more, and I've got the point I would like to talk to others who have tried successfully or not to do this process and learn everything else I can. Hey NebulaHaze you seem to be very well versed in a lot of topics and filled with lots of full and complete answers, I'd love to hear anything you have to say on this subject.

[growmore]

Hi Greenthumb
About 20 years ago I owned a tissue culture lab growing 200,000u plants a year. Tissue culture is mainly used to mass produce identical plants of specie's that are either slow growing or hard to propagate. Mj plants are easy to strike from cuttings and fast growing, so unless you wanted to grow large numbers very quickly it would de simpler to grow by ccutting.

[PigSquishy]

Actually my interest in it is now just for cannabis, I was actually looking at it as a way to get a lot of other plants going and working to get more food crops going. All it all it seems like a perfect way to get a leaf from a tree, plant, etc... and then turn it into 50 plants of the same plant, all clones in essence of the same mother. I've read you can do this to keep mother's alive and taking up very little room. I've wondered if I can't apply this to food crops growing inside and outside as well? I have a lot of questions on this topic, and not all relating to cannabis.

[growmore]

If you have any specific questions feel free to ask

[Komatchi]

Do you have your TC protocol for mmj still?

[growmore]

I have never grown mj in culture. I used to grow
Australian natives and gerbera's. Would suggest trying 1/2 strength ms medium, 30 grams sucrose and 8 grams agar per litre as a starting point. 0.2 mg litre bap for multiplication and 2 mg iba for rooting.if you are starting with meristem pieces. I would not recommend growing from Leaf material as you are likely to get genetic differences.

[NebulaHaze]

It's hard to follow after growmore's personal knowledge and experience in this subject!

I agree that part of why cannabis growers don't do it is it's difficult compared to cloning cannabis the "normal" way, which is really easy and doesn't need any special equipment. You can clone a cannabis plant with just a pair of scissors and a glass of water. If you cut large clones, you can get 7-10" plants growing in just 1-2 weeks in good conditions! I imagine tissue culture cloning takes longer to get plants that size even in the best conditions.

But living tissue culture has some cool applications that might be useful to serious cannabis breeders. It can be used to help propagate a particular plant that has been infected with a virus (for example maybe you have a great plant that somehow caught TMV but still want to make clones).

Tissue culture cloning can also be used to cross-pollinate distantly related species that normally couldn't breed, creating novel hybrid plants. I wonder if that has interesting applications down the road. What kind of plants could you combine with cannabis plants to alter their effects, or how could you influence other plants by combining them with cannabis?

One thing that comes to mind is the hop plant (the main ingredient in beer). Both cannabis and hops are in the Cannabaceae family (relatively closely related), and both are grown for their female flowers... What kind of interesting hybrids could be developed from the two?

[cyberaktif]

weed beer !!! LOL

[growmore]

You can use tissue culture to propagate almost anything. As for not taking up much space commercial labs run about 140 plants per square foot in racks with shelves every 10 inchs.

[PigSquishy]

Thank You NebulaHaze, I must admit it doesn't surprise me all that much that you caught on to exactly what I am up too... I have been looking at grafting two strains of cannabis together, because it will affect the genetics of the two halfs of the plant, and from that you can end up breaking it down and instead of trying to breed its qualities out in seeds, you can clone the mother. Now from what I've been reading on this topic, and please growmore please correct me if I misunderstood what I've been reading on this subject. But it is my understanding that it would allow you to even back track the genetics a little bit by making the genetics stronger by doing the leaf tissue culture, something you can't do in clones. If I understood what I read correctly you can get better genetics for mothers by doing this process and combined with selective breeding can really come up with some amazing hybrids.

Hops isn't the only question I want to play around with relating to cannabis, but also the tomato plant as well... I would love to know how cannabis would do in something like companion gardening, two books I have on that subject to explain what this is are "Carrots Love Tomatoes" and "Garlic Love Roses".

Not to mention I just can't get over what this could mean for my flowers and gardens at the end of the day... How many flowers, fruit trees, bushes, etc... could I really grow and be able to bring to farmers market every year? Perhaps it would be cost productive to be able to grow more plants, trees, etc.. to bring to market than the fruits? No matter how it works at the end of the day it's like this... Not far from me they built a cannabis cultivation place to grow it, and they were suppose to open and then hit a snag of an issue preventing it from opening so these big named people they had to come run it backed out. They made it know they would be interested in hiring people later on who know about and their was a list of things, tissue culturing being one of the things on the list, so I want to make sure I am well versed in it and how to do it properly. At the way things continue to change I guess you never know when the job I apply for next is going to ask if I know how to do it and I want to make sure I am well educated in how to do it properly according to all safety procedures.

So please by all means I am seriously willing to sit down and do whatever countless hours of reading I need to do, watch however endless hours of YouTube videos I need to endure, and even spend some cash to make the DIY Laminar Flow Hood or just spend the money and buy one right off Ebay. Please understand I am truly serious about wanting to learn everything I need to be able to do this, I want to research it, learn it and be able to do it and not just be limited to just the cannabis plant either.

[DingusKhan]

Based on your objective, it sounds like what need to do is enroll in college with a major in botany, emphasis on cellular biology. The expertise required to grow individual cells in a dish into multicellular organisms is the some PhD level shit, that as Growmore said takes place in a sterile lab with insane precision.

I think you should go for it.

[growmore]

Tissue culturing any plant will not change it's genetic makeup unless you use a mutagenic chemical to achieve that.. As for breeding new strains, the only way to do that is by culturing haploid pollen grains and using a method called protoplast fusion which is what nebula was referring too. By diong that, you can make a strain that will breed true All the plants generated by that hybrid will be slightly different and you would then have to grow them to maturity and select the best plants

[PigSquishy]

Well I do honestly and sincerely understand the mountain of a task before me in trying to undertake this, going to college to learn it all isn't an option for me, but none the less it doesn't mean my final education on this subject will be anything less than a college education on the subject at hand. I do not really know how to sum this up and still get the serious extent of my point across here, but I am going to try to... Despite not going through the years of proper, formal college education I have many times over proven I am just as or more qualified for the same jobs as those with the formal and proper education according to the degree on the wall; you can not begin to imagine how hard it is to have to prove yourself more qualified for something without a stupid piece of paper, but it hasn't stopped me from getting to the places I've wanted to get to and had exceptions made for me plenty of times. There hasn't ever been something that has interested me, peaked my interest or that I've wanted to know more about in depth that I haven't dedicated the time and research into studying until I can understand it on the proper depths as scale to do it correctly with proper safety precautions.

Now thus far I've been checking out "home tissue culture kits" enough to get an idea of the basics of what all comes in the kit and start trying to get a basic concept of what all is involved, I've taken the time to check out some companies who want you to buy their kit showing you how easy it is to do it at home yourself. I have been speaking with Harley Smith, a man with a Ph.D who holds lectures at NPK University, works for NPK Industries, and has in my opinion a very impressive stellar reputation in the hydroponics and scientific communities for his work (not in cannabis). He and his team have also done studies for world governments, thus you know that his work is real and can be trusted. He has also written articles published in Maximum Yield; and has really changed the power balance between the grower and the nutrient companies, by putting the power back into the growers hands with his work with NPK Industries. He told me to get the book ""Plants from Test Tubes: an Introduction to Micropropagation" by Lydiane Kyte and John Kleyn.

I understand to the average person what I am trying to undertake is far above the scope of what most growers would ever want to, but this new home tissue culture kits are showing up in hydroponic shops and aren't super expensive either, so I am sure it is something that will catch on. My Great-Great Grandma came from Germany and brought seeds with her on the boat, sewn into her belt she used to tie her dress around her waist, she would plant those seeds in Surrey, North Dakota and be the only place in North America those plants can be found, according to the Minot University professor of horticulture who takes his students out there to study them with his students says those plants shouldn't grow where they do. My grandma moved a "Flowering Cabbage" flower which can't grow outside of 30-miles range of the Gulf of Mexico locals claim and grew it in North Dakota. My grandma even made the first ever Black Iris according to the local nursery who couldn't understand how she managed to pull it off, and at the end of the day none of them had any college education or any formal teaching to show them how to do it. My point is this, I am 100% serious about learning, studying, researching and moving in a forward direction with this... I will not be easily turned away, nor will I give up when I find its hard and its not super easy, but takes a lot of time of actually sitting down to do precision work.

As it stands right now I already take care of various fruit bearing trees and bushes, I raise and care for a garden of food crops both above and below the ground, and then I also raise flower gardens for bees and butterflies, and despite all of my time and experience as a soil grower, I am also familiar with hydroponics and feed my various fruit bearing trees and bushes on a passive hydroponic system while other zones are on active hydroponic systems. Since childhood I have longed to design, build and perhaps even one day bring to market a hydroponic system to fit inside your kitchen so you grow your own food, and don't have to store it in can goods. When I came to start studying and learning about growing cannabis I only did so back then because of academic reasonings and it transformed an amazing amount of things for me, I was finally able to learn to do even more with gardening and plants because of what I learned. Honestly I am hoping that Leaf Tissue Culture is going to help me as another stepping stone in my long term quests and goals relating to hydroponics and being able to make it so everyone can grow their own food and medicines within the comforts of their own homes. At very least I am hoping I could take clones of the fruit bearing things I have and be able to sell or give them away to others with the hopes of increasing the local market on locally grown fruits. There are also things like the Prairie Radish that you can sometimes just find one of and that's it, and if I could cut up the leaves to make more explants and still get to enjoy the radish, well win win... I am not trying to limit myself to the possibilities of what you might be able to do with such a special thing, like maybe even grow more Diamond Widow trees which are serious endangered... I really do sincerely want to look much deeper into what all I can do with this and learn the real costs and benefits of everything I can and can't really do with such technologies.

[bobsakamoto]

funny story...one of the best friends i ever had was a new-to-me mechanic i took my car to for a simple repair; he was rec by a friend. the insides of my car were all removed because that was a city that routinely flooded in like 45 min so my first month living there my car got flooded while parked. so i had to take out all the interior, seats included, all the carpet padding, all of it down to the metal...then suck out all the water any way possible, then baking soda it to prevent future smells. anyway, he took one look at the interior and was like "what happened here?" i told him. and he was all like, wait, what? because naturally since i'm a girl, i could not have done such a thing....much less be seemingly doing it in exactly the manner in which it should be done in order to be fully successful... we became bffs that day.

[PigSquishy]

Yeah I actually had to teach a girl I know outside of school auto mechanics a few years back because the school wouldn't let her into automotive class, because "she was a girl and girls don't take that class". So while she classmates worked on engines, we rebuilt an engine together that was hers and she knew everything in detail about, which only makes it all the funnier to have a sleeper "semi-street legal" truck on the streets. Nobody took her seriously until they heard the pipes when the clutch came out and it took off... never underestimate what someone can do when they are told they can't...

[bobsakamoto]

that's a great story

[growmore]

I know nothing about the kits you are referring to, if possible post a link so I can see what they are saying you need? Have you read the book you referred too.

[DingusKhan]

The real benefit of applying this level of technology to growing plants is in producing quantities sufficient to end food shortages in poor countries. How many plants do you intend to grow?

At the same time, stoner-doods with scissors and loud music can already produce warehouses of clones, more than they will ever need. I myself have created a bunch of clones, and I just started growing pot for the first time 2 months ago using a costco rotisserie chicken container.

I find this thread a welcome addition to a forum devoted to "growweedeasy". In addition to the many sages sharing their wisdom, it sounds like there are a few people with advanced degrees in completely unrelated fields, and some people who don't know what pH is, looking for help for their dying plant. I myself am doing silly shit like cloning with a rotisserie chicken container.

What a forum!

[growmore]

The end results are all that matter. If something works for you then do it. It's not about how you achieve things but the results.

[bobsakamoto]

if there is *ONE* benefit to having this particular plant being precluded from serious, double-blind, peer-reviewed, the whole ball o'wax, so to speak, STUDY by the scientific community it might be this^^^ that whatever works works.... and there is no "governing body" to tell you, the individual "scientist", otherwise.

[DingusKhan]

True that, Bob.

[PigSquishy]

Well I have at this point got the book on order from Amazon, but then I heard it won't ship off right away... so I haven't had the chance or opportunity to read it yet, just everything I can find doing searches and digging into anything they say I don't already know or understand. I've put together a basic list of somethings thus far, it will perhaps give you some idea of how much I know and where I am at thus far.

-Tween 20
- Sodium Hypochlorite (Bleach)
- Laminar Flow Hood
- Agar
- Magenta Vessel with a gas permeable lid
- T-5 Nano Tech By Sunblaster for lighting
- (Plus the basics such as sterile scalpels, gloves, lab coat, etc...)

The kit I spoke I found here:
http://growershouse.com/microclone-s...ue-culture-kit Kit includes:

• 50 6oz flip-top vials
• 30 clear plastic jar lids
• 16 starter poly test tubes with screw lids in tray
• 5ml PPM antifungal/antibacterial preservative
• 1 establish/multiplication media kit with BA hormone
• 1 rooting/growth media kits with NAA hormone
• 2 establish/multiplication TDZ media kits with TDZ hormone for branching and woody plants
• 1 stainless steel scalpel #3 and 2 #11 blades
• 8-inch stainless steel forceps
• PVC sealing tape
• pH control kit with test papers and correction solutions
• 50-page instructional manual with color photos and illustrations
• DVD instruction video
• Washing screen and ring for standard mouth jars

As long as I am going through all the work to learn and do this, I also considered being able to use this same set up for mushroom growing, it has long been something I'm interested in as and this same equipment is needed to do that as well. In another post I even brought up I have even found the DEC-4 by Sentential which is an Environmental Controller which controls, Heat - AC - Humidity and CO2 (1100-1300 ppms) and has an on at dark cycle for mushroom growers unlike a lot of other environmental controllers. Comes with a 5-year warranty and is only $280 compared to a basic CO2 controller which does nothing else starting at $200.

DingusKhan Yeah I know exactly what you mean, I have been surfing Forums for awhile looking for educated people to have conversations with beyond the basics of pH and nute issues... I mean I love helping new growers get going and get it right, but at the same time I want to be able to hold intellectual conversations about other stuff too, and I am over joyed to have found this Forum with such helpful people who are also very knowledgable as well. I have grown plants in whatever I have been able to find as well, at one point the inside of my house was a living garden all being run on different forms of hydroponic systems for over 2-years until... well I asked a couple friends to watch my gardens.

[DingusKhan]

That is a painful, hysterically funny story. I'm still in my very first grow which is in fact my introduction to gardening in general, and I think I've gotten the bug, meaning I LOVE it (although I'm pretty sure I will limit myself to one room). Which means I will probably need friends come and plant sit too.

What's really funny is that my primary motivation for growing and joining this forum is to escape from my biology-based profession. I should be avoiding threads about cell culture! But now I'm hooked and excited to see what you do with this. And even though cell and molecular biology is only loosely related to what I do, I hope there may be some weird tidbit of knowledge stashed away that I may share, even if it pales in comparison to what growmore has.

Godspeed!

[PigSquishy]

Well a word of advice if you ever need someone to come plant sit... DON'T EVER ASK ANYONE!!! I will spare you what happened to me, but just sum it up to say it cost me 5-years scrubbing mold. The 2nd time I came home to NOTHING, my "friend" of many long years took even the curtains and toilet paper along with everything else and then to my disbelief couldn't even figure out why all the plants died and why he couldn't make it work since he had all my stuff, and knew everything I told him to do for them.

[growmore]

Greenthumb Ok I looked at that website and from what I can see they sell the culture kits but don't specify what the media is or the concentrations of the hormones and when I searched for nutrient media refills it came up as no items found. I can see no point for buying this kit, apart maybe for the manual and disk, as you can easily get online from a lab supply company for glassware and instruments and Sigma chemicals used to publish a catalogue specifically for tissue culture.

Other things you will need are

Pressure cooker (15 PSI )
Bunsen burner ( for flaming scalpels and forceps)
Ethanol 80% V/V (for sterilizing forceps scalpels inside of flow cabinet)
Distilled water (Water still or RO unit)
Glass petri dishes (for cutting up explants)
Volumetric flask and graduated pipets for measuring fluids
Alfoil (for wrapping scalpels forceps petri dishes before you sterilize them)
Culture vessels ( I used to use a 30 ml flat bottomed polycarbonate tubes for starting off new cultures and once established then use a round Polycarbonate screw top container 70 mm wide and 80mm high. Brand name BUNZL)

On your list you mention a Magenta vessel? Can you explain what that is as I have never heard of that before.

Hope this helps