Tissue Culture / Micropropagation

Hey Blades,

I decided to share my interest and expertise in the field of horticulture that I specialize in, which is plant tissue culture and micropropagation.

*Picture of some African Violet plantlets I started a few weeks ago*

Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. Plant tissue culture is widely used to produce clones of a plant in a method known as micropropagation. The tissue culture starting material can be as small as a single plant cell, because plants exhibit totipotency, or the ability to regrow a completely new plant from a single somatic cell, but the most common explant material is leaf tissue or tissue from a meristematic region such as the apical meristem.

Micropropagation is the practice of rapidly multiplying stock plant material to produce a large number of progeny plants using tissue culture methods. Once a tissue culture has been established and plantlets start to form, you can then theoretically derive an exponential amount of new plant material from the starting material.

Micropropagation Steps:

1.) Establishment - Micropropagation begins with the selection of plant material to be propagated. Clean stock materials that are free of viruses and fungi are important in the production of the healthiest plants. Once the plant material is chosen for culture, the collection of explant(s) begins and is dependent on the type of tissue to be used; including stem tips, anthers, petals, pollen and others plant tissues. The explant material is then surface sterilized, usually in multiple courses of bleach and alcohol washes and finally rinsed in sterilized water. This small portion of plant tissue, sometimes only a single cell, is placed on a growth medium, typically containing sucrose as an energy source and one or more plant growth regulators (plant hormones). Usually the medium is thickened with agar to create a gel which supports the explant during growth. The plant tissue grows and differentiates into new tissues depending on the medium. For example, media containing cytokinins are used to create branched shoots from plant buds.

*Some watermelon cultures I started with tissue from the seed embryo*

*Media contains a cytokinin 6-Benzylaminopurine to induce shoot formation*

2.) Multiplication - Multiplication is the taking of tissue samples produced during the first stage and increasing their number. Following the successful introduction and growth of plant tissue, the establishment stage is followed by multiplication. Through repeated cycles of this process, a single explant sample may be increased from one to hundreds or thousands of plants. Depending on the type of tissue grown, multiplication can involve different methods and media. If the plant material grown is callus tissue, it can be placed in a blender and cut into smaller pieces and recultured on the same type of culture medium to grow more callus tissue. If the tissue is grown as small plants called plantlets, hormones are often added that cause the plantlets to produce many small offshoots that can be removed and recultured.

*African violets started a few months ago in one jar, now many jars*

3.) Pre-Transplant / Hardening off - This stage involves treating the plantlets/shoots produced to encourage root growth and "hardening." It is performed in vitro, or in a sterile "test tube" environment.
"Hardening" refers to the preparation of the plants for a natural growth environment. Until this stage, the plantlets have been grown in "ideal" conditions, designed to encourage rapid growth. Due to lack of necessity, the plants are likely to be highly susceptible to disease and often do not have fully functional dermal coverings and will be inefficient in their use of water and energy. In vitro conditions are high in humidity and plants grown under these condition do not form a working cuticle and stomata that keep the plant from drying out, when taken out of culture the plantlets need time to adjust to more natural environmental conditions. Hardening typically involves slowly weaning the plantlets from a high-humidity, low light, warm environment to what would be considered a normal growth environment for the species in question. This is done by moving the plants to a location high in humidity.

4.) Transfer From Culture - In the final stage of plant micropropagation, the plantlets are removed from the plant media and transferred to soil, hydroponic, or (more commonly) soiless media for continued growth by conventional methods.
This stage is often combined with the "pretransplant" stage.

So, how do we apply this to our favorite plant, cannabis? Unfortunately there isn't anywhere yet in the US that the ability to mass multiply cannabis would be needed, because each cell culture could potentially be considered a cannabis plant on its own and the vast number of plant material that could be created would surpass the allowable plant limits under most MMJ laws. I could be wrong on this, please chime in if you have a more definite answer.

 

Edit (5/18/2013):  The previous paragraph is no longer the case with the legalization of recreational marijuana use and sales in Washington and Colorado.  I am only familiar with the particulars of the current Colorado laws and regulations, and it looks like a full fledged commercial cannabis tissue culture and micropropagation lab would be legal. 

One possible application for this technique could be the storage of a large selection of different strains to be multiplied when needed and yet not taking up as much space as maintaining the same number of mother plants.

I'm planning a few experiments right now to test which media mix would be best for each stage of the micropropagation process with cannabis. So far the only reference I could find to give me a place to start was here:

http://www.pakbs.org...PJB41(2)603.pdf

This study was for the micropropagation of Hemp grade Cannabis Sativa, however, so from my experience with different varieties of watermelons, the concentrations of hormones will probably be very different for Recreational Cannabis, but the particular hormones stated TDZ and NAA will be a good place to start.

Well, wish me luck and let me know if any of you have questions pertaining to this exciting side of horticulture.

Edited by Berry, 18 May 2013 - 01:46 PM.

This is my next quest, looks very interesting

Thanks for the informational thread. Its nice to see a true advance growing article in the advanced section. I read an article on this a few months back. Sounds so awesome for those of us that search and Search for the "perfect" pheno. Sounds like the perfect way to preserve good phenos. *sigh* no room Or cash to do something like this.

Thank you for the post I have been looking in to this more and more. I will be starting up in the next few months. I will follow and pick your brain randomly if you dont mind?

So from what I gathered, you could have a mother that is basically a tiny ass plant inside of a jar?

That sounds pretty fantastic.

This is something I'd probably partake it. It'd be great for a sog. You wouldn't really need veg space. Once you've got a seedling, just throw it into flower...

I'm going to keep an eye on this thread, this may be something... worth it.

Thank you for the post I have been looking in to this more and more. I will be starting up in the next few months. I will follow and pick your brain randomly if you dont mind?

Pick away, lol. I've been specializing in this for over a year at college. I have access to a pretty nice lab with all the chemicals and hormones I could ever need to do experiments, so it has been pretty fun. If I don't know the answer I can just ask my professor.

So from what I gathered, you could have a mother that is basically a tiny ass plant inside of a jar?

That sounds pretty fantastic.

This is something I'd probably partake it. It'd be great for a sog. You wouldn't really need veg space. Once you've got a seedling, just throw it into flower...

I'm going to keep an eye on this thread, this may be something... worth it.

Yeah, you can pretty much store 100's of motherplants in jars, in the same amount of space it would take to veg one mother plant, and with a fraction of the energy cost because the plants in the jars don't need much light because they get their energy from sucrose you mix into the gel media.

That is probably the biggest advantage to using this technique, so many different strains in a much smaller space. You'd want to have more than one jar for each though because of the chance of contamination.

Thats one thing I didn't cover very much in my first post. Contamination is the biggest problem, everything has to be kept completely sterile because bacteria and fungi will grow faster than the plant cells on the gel media and fully contaminate a jar in a couple of days. You don't necessarily have to use an expensive transfer hood and autoclave ect. You can get by with homemade clean boxes like aquariums tipped over on their side with clear plastic covering the opening, and using pressure cookers or even a microwave to sterilize your media.

It's definitely interesting. It'd be nice to have a setup like this.

Personally, I'd find most use from the fact that they can be reproduced until you have a ton of clones so to speak. That would be excellent. At that point, I could probably get rid of a veg room all together, and just veg for a week or two before flowering a bunch of small plants.

Very interesting stuff. The breeding aspect is very interesting too. Who wouldn't want a plant that'd produce huge yields and a great high?

Do you have any links to good kits or do you recommend any specific type of kit/
And what about hormones?

Id just like a compact way to preserve strains long term over the course of years. One tray with say my 10 all time fav of a particular strain that I know are female.

Do you have any links to good kits or do you recommend any specific type of kit/
And what about hormones?

Kits would be good for just getting the basic supplies you'd need, but most of them will include hormones and media mixes that are tailored for a particular plant that the kit is to be used for; african violets, hosta, and orchids being the most common. The african violet kits will probably be the best. Just remember that the hormones that would be needed to propagate cannabis, and the media mix needed will almost definitively not be the same as for african violets.

The general mix that we use in the lab to start off with when we are trying to develop a propagation protocol for a plant that doesn't already have a protocol established that we can reference to find the best mix of hormones and media salts is:

Basic Media Mix
*These amounts are for a 1000ml Batch, so adjust accordingly for smaller batches, 10-20ml is usually used per jar/tube*

- 3g Gelling Agent:I prefer gellan gum, because it turns out perfectly clear which helps with identifying when you have contamination.

- 4.5g Murashige & Skoog (MS) Basal Salt Mixture: Product ID=524, there are alot of variations of Nutrient salt mixtures out there, but this is the best basic one to start with.

- 30g Sucrose: off the shelf table sugar from the supermarket works.

- Distilled Water: start off with less than the end amount, like 800ml for example, then top it off once you've added media components.

- Hormones: the hormone concentrations will vary and are usually given in ppm or mg/L (1ppm=1mg/L), so the liquid hormones that are usually 1mg/ml are useful for correctly measuring out hormones.

I do all my media mixing in the laboratory in an erlenmeyer flask on top of a hotplate/stirrer. Always use a flask that is twice the volume of what you are mixing in it, so a 2000ml flask for a 1000ml batch. You heat the distilled water up, but always keep below boiling. Then mix in media components, with gelling agent last because it will thicken up the mixture, then add distilled water to bring the mixture up the volume needed. Next pH adjust the mixture, we use pH 5.7 for most of our media mixtures but cannabis might like it a little higher, I'm not sure yet. Add your hormones last, then you bring the heat up until the mixture turns from cloudy to clear and just starts to bubble, then you remove it from the hotplate and syringe off 10-20ml per jar/tube. You put the cap on the jars and then either autoclave or pressure cook to sterilize. The important thing is to never take the lids off outside of a clean box/transfer hood or the media will become contaminated by bacteria or fungi. Jars of media can be stored in the fridge for later use.

We use this company for hormones and media mix components alot: Home Page - PhytoTechnology
They also sell almost anything else you would need to get started including lab equipment, utensils, and some kits for propagating hosta, african violets, and orchids.
They have alot of useful references on their technical info page:
Technical Info - PhytoTechnology

Another good source for kits and information, especially for low-budget home micropropagation is:
Home Tissue Culture  Group
They have less of a selection, but they have alot of useful links for how to get started doing tissue culture at home.

A good book to read for some more information would be:
[ame="http://www.amazon.com/Plants-Test-Tubes-Introduction-Micropropagation/dp/0881923613/ref=sr_1_1?ie=UTF8&qid=1329174101&sr=8-1"]Plants from Test Tubes: An Introduction to Micropropagation[/ame]
Which I also just found on piratebay so you can check it out before you buy it if you want.

This obviously is a lot larger than most of us 'hobby' growers would probably take on.

I think it'd be wise, and probably pose monetary gains for you, if it'd be possible to sell the mixes for cannabis micropropagation. There'd be nothing really illegal on your part, with the exception of determining the hormone levels.

It's very interesting, but overwhelming simultaneously. A 'kit' for cannabis would be awesome. I'd most definitely invest 300-400 dollars if it'd last me to make at LEAST 300-400 clones. It'd be so nice to have something that only took up a few feet of table space, but provided me with so many clones, that'd be a step for me and growing. My output would definitely increase.

This obviously is a lot larger than most of us 'hobby' growers would probably take on.

I think it'd be wise, and probably pose monetary gains for you, if it'd be possible to sell the mixes for cannabis micropropagation. There'd be nothing really illegal on your part, with the exception of determining the hormone levels.

It's very interesting, but overwhelming simultaneously. A 'kit' for cannabis would be awesome. I'd most definitely invest 300-400 dollars if it'd last me to make at LEAST 300-400 clones. It'd be so nice to have something that only took up a few feet of table space, but provided me with so many clones, that'd be a step for me and growing. My output would definitely increase.

Yeah I totally understand how it can seem overwhelming, honestly it has taken me 2-3 years of study in horticultural science to develop a knowledge background to where I fully understand how to successfully do the experiments to determine what hormone and media mixtures that would be necessary for growing cannabis with this method.

This is the field I plan on going into once I graduate in May, and after that I'm moving to Colorado and plan on trying to develop ways to bring these techniques to the MMJ industry. I was thinking about setting up a website with kits for sale with hormones and media mixes tailored to the growing of cannabis. Another thing that I am looking forward to is that once I develop a protocol for growing cannabis this way, the procedure and homone/media mixes will be slightly different for various strains. So there would be a different kit for growing sativa dominant strains and indica dominant strains.

So far I haven't really seen many online hydro stores or grow shops selling much in the area of micropropagation. I've seen some kits, but they are very generic, and wouldn't really be that useful.

With the right kit, anybody could do this really. The hard part is figuring out the right hormone concentrations and media mix to use, and also the best part of the plant to use as explant ect.

Thank you for the info. I agree with ohspyro89 I would be willing to pay $200 -$300 for a MJ kit. Shot berry something you should think about.

I'd pay that just to be able to collect strains

Very interesting, I've been looking into the same technique for mushroom cultures, ill definitely be watching this

Very interesting, I've been looking into the same technique for mushroom cultures, ill definitely be watching this

There was actually a guy doing some mushroom propagation the other day in the the same lab I was doing my micropropagation. Seems like it is very similar.

Right now I'm just waiting on some seeds to germinate. I started some random 2 year old bag seed I had sitting around a few days ago. I figured they would be good for practicing with different hormone and media concentrations. Only 2 out of 5 germinated and neither have actually sprouted so If I don't have some seedlings come up by next week I think I'll germinate some Blue Mystic feminized seeds I have left from my last grow.

I don't have a full fledged grow going at the moment because I'm renting a townhouse from the in-laws and we are about to start doing some renovations in about a month so they will probably be dropping by to see how things are going more often than the come by now.

I'll probably just veg a plant out for a few weeks, then take lots of tissue samples from different parts of it to start a bunch of tissue cultures. Then multiply those out until I get enough plant material to start transferring them onto different media with various hormones to see what works the best.

That calls for a PC grow no? There's quite the cult around here for it...

and your full fledged grow would be what ? 6 plants in a 1x1 inch area?

you could do a micro-field of green ha ha

have you dabbled with natural hormones? like mannitol or salicylic acid?

thanks for the amazing guide Berry, I'll have to read through it again....

hey berry I was thinking about this post and remembered that Highttimes had a article on tissue culturing I think I may have that article I will post what issue it is it may help

So I just found a site that looks like it has done the study already. They have published it at this site http://www.pakbs.org...PJB41(2)603.pdf . I hope this is a help please let me know.