Cannabis tissue culture

[jimbop]

But what about for having a library of strains? For example if I wanted to have 25 strains on standby, without keeping mothers. Could I keep these in culture in a relatively stable fashion, and then divide and root the callous tissue into new starts as needed? How long do you think it would take to go from callous tissue to a hardened start suitable for planting?

And for the purposes of a library, how long will a sterile callous culture last? Months? Years? What is the maintenance like?

I'm also interested in those cultured seeds as well

Callus culture is not a good way to do this, mainly because if you have some form of contamination, you've lost your lines. The solution would be to have multiple containers of each line, but even then there is the chance of loss- not to mention all the extra work it takes to go from singlet to triplicate (or more). All it takes is an infestation of mites, and you'll be autoclaving hundreds of containers with each daily rouge, as any lab or any reasonable size has experienced at least once. Maintenance over the course of years or decades becomes risky and expensive- a bad combination. Normally DMSO + cryogenic storage (liquid nitrogen) is the best answer, a trivial problem for any well-equipped tissue culture lab.

Even then, I remain unconvinced as to the use of "libraries." Marijuana growers, as a group, are very forward-thinking: who cares where we've been? If the old strains are so good, why aren't they more popular? Everyone wants the next best thing, not last weeks' hot topics. Go through any book on marijuana that's more than 5 years old, and try to find cuttings or seeds of any of the old favorites; you will find it difficult to locate the vast majority of these.

I don't know what you mean by cultured seeds. Do you mean embryo rescue, or perhaps artificial seeds?

 

[ganjourno]

Yes artificial seeds is what I meant - sorry it's been a few years since I messed with TC. Sounds like artificial seeds would be a good middle road to saving some strains. My need for a library is not necessarily to have an archive of every strain ever, but let's say every killer phenotype, to be able to preserve it for future use in a semi robust way, that is just not practical to do with live mothers. If I could make a few artificial seeds and save a strain that way, and be able to germ those seeds within a few years time frame, would be extremely advantageous. Would they keep in a normal freezer for a few years (provide they were vacuum sealed without moisture)?

I addition to the artificial seeds, could you recommend a basic explant preparation routine, in terms of initial hormones, sterilants, and high level process?

Thanks for your help

 

[Only Ornamental]

So after your comments, my failure was likely due to the sterilant I used (90% alcohol). Not necessarily or rather likely not only.

... All the equipment and water was sterilized in a pressure cooker prior to use... How long? Besides, chances are very high that you're doing it wrong (no offence, just experience).

The medium was an agar mix with pre-weighed nutrient salts froma tissue culture supply place, mixed and poured into tubes then pressure cooked for a while. What the heck is A WHILE?

...The tubes were sealed with some thin strips of cling wrap. Which supports my above claim that you're doing it wrong (the pressure cooker thing). Besides, cling wrap is clean but not sterile. Why not use some sort of lid you can sterilise/pressure cook as well (like a rubber plug with cotton filled hole)?

So it seems like the alcohol is where I went wrong. After 1-2 weeks most of my cultures developed moulds.

Small advice: If you start, do a dry run and get some routine. Then use a cheap culture medium replacement whereupon microbes should be able to grow, such as fruit juice or other organic matter, and simply pressure cook them and incubate. Next, do the same and 'mimic' adding explants but just do the 'empty' handling and incubate again. Build the thing up step by step cause only that way will you be able to figure out where you do mistakes and from where the contaminations come from.

You shouldn't leap directly to artificial seeds but start small, one step at a time.

 

[jimbop]

If I could make a few artificial seeds and save a strain that way, and be able to germ those seeds within a few years time frame, would be extremely advantageous. Would they keep in a normal freezer for a few years (provide they were vacuum sealed without moisture)?

I can't speak to that. I 've only kept germplasm at -80C or in liquid nitrogen vapor. Never tried commercial refrigerators, which are typically -20C.

Synthetic seeds aren't always capable of being stored in the fashion you are thinking about. I don't know as anyone has developed a protocol for desiccated synthetic seeds, nor those that can be frozen.

I addition to the artificial seeds, could you recommend a basic explant preparation routine, in terms of initial hormones, sterilants, and high level process?

Explants:

Depends upon whether I'm working with nodes or leaves. Generally I use a sterile container- sterile 50 ml centrifuge tubes are good for nodes, an autoclaved glass container good for leaves. Leaves and nodes of outdoor plants should be washed, debrided, maybe be given a quick detergent wash if you want. Indoor material generally has a lower biological burden.

Bleach is the generally accepted disinfectant; however, some folks I know have success with hydrogen peroxide if the tissues are bleach-sensitive. Peroxide works best when exposed to light, but the containers must be vented; 50 ml centrifuge tubes will rupture.

Newer concentrations of bleach make it so that some math is required to get the ~0.5% sodium hypochlorite; used to be they were all 5.25% sodium hypochlorite, now it's 6% and up. Use bleach without any fragrances, detergents, etc.

I prepare a liter of disinfectant at a time; I add in ~0.5 mL Tween (Tween 20, Tween 80, whatever's handy- Palmolive works fine). Disinfect with occasional agitation for 5-30 minutes, depending upon how difficult I think the tissues will be to disinfect. Ensure all parts of the glass and stopper are evenly exposed to sterilizing solution. Wash once with sterile distilled water. Embed in media (nodes) or lay on surface (leaves) abaxial side up. Repeat until all tissues are prepared. I'll generally knock back 10-25 leaves or nodes in one sitting in order to assure one clean culture at a minimum.

Details as to media and so forth are a bit involved- and, for the most part, proprietary. As gleaned from notoriously unreliable Chinese publications, the literature values are close enough to get most people started. Some optimization will be required depending upon culture conditions and the cultivar being maintained.

 

[IntelliGeneS]

agrobacterium... taking multiple gene from different strain and cross genetic... take some of your favorites strains, make callus from it, use a bacteria to infest the callus and absorbs its trait by infestation for a day or two, then culture tho gene imprinted bacteria, propagate them into a new strain callus, as it infect the new strain it will also incorporate it'S gene, then kill the bacteria with it's antibiotic, and you'll have your self a new strain, if on where to have a libraries of most plant families and species on hand, one could someday make strawberries high in thc, and that would be the end of all illigal issues related to cannabis, since thc could be found in all living plant... (of topic) but hey, that was my dream when a was young, have on hand over 2500 strain of cannabis and make high in thc fruit, tho i do not consume cannabis' i still love fruit and consider myself a cannabis passionate

No offense intended, but you have only a barely surface level understanding of Agrobacterium-mediated plant transformation.

While optimizing Cannabis callus growth in vitro and plantlet regeneration protocols are significant undertakings in themselves, designing/cloning/preparing a gene insertion cassete for Agrobacterium-mediated plant transformation would be another thing entirely. While tissue culture makes plant transformation possible, it is out of scope for this discussion. If you would like to discuss more about transformation and direct genetic transfer or manipulation techniques, please start a new topic... however be prepared for the thread to devolve into knee-jerk anti-Monsanto scapegoatism and catastrophic fear responses, which are inevitable.

What would be very interesting to me is if anyone has had success generating haploid plantlets or callus of Cannabis via anther/ovum culture or similar methods. This would be a step forward in tool development to help start to resolve the current "pheno-hunt" business methodology in Cannabis breeding which results in a highly inconsistent product being widely sold. DH breeding might be a powerful strategy to pursue.

 

[jimbop]

While optimizing Cannabis callus growth in vitro and plantlet regeneration protocols are significant undertakings in themselves, designing/cloning/preparing a gene insertion cassete for Agrobacterium-mediated plant transformation would be another thing entirely.

Moreover, I have yet to find a concise explanation as to what transgenic cannabis crop would be valuable anyway. Herbicide resistance? Maybe for hemp; that's about it. Higher levels of specific cannabinoids or terpenes? Much easier through selective breeding.

 

[IntelliGeneS]

Moreover, I have yet to find a concise explanation as to what transgenic cannabis crop would be valuable anyway. Herbicide resistance? Maybe for hemp; that's about it. Higher levels of specific cannabinoids or terpenes? Much easier through selective breeding.

Agreed, there's not much reason for wanting trans genetic transfer into Cannabis. I imagine the most likely applications would be virus resistance or possibly PM/mite resistance (which are not currently feasible). Otherwise as a vegetative crop specifically for human consumption, transgenics is a non-starter, just as it is for other crops in that class (except sweet corn, for historical and familial reasons).

However, tissue culture would be very valuable for many reasons, just not so much for genetic preservation (plants already have a more efficient mechanism for that: seeds) or GMO work.

 

[anthonymalich]

the guy in mt view

the guy in mt view

Not sure what company you are using, but I know the guy that makes these out of Mountain View California. This guy really knows his shit and makes a great product.

I met the guy in mt view a really long time ago. I how do I get in touch with him or order one of his kits? Please email me at [email protected]

 

[Sam_Skunkman]

Agreed, there's not much reason for wanting trans genetic transfer into Cannabis. I imagine the most likely applications would be virus resistance or possibly PM/mite resistance (which are not currently feasible). Otherwise as a vegetative crop specifically for human consumption, transgenics is a non-starter, just as it is for other crops in that class (except sweet corn, for historical and familial reasons).

However, tissue culture would be very valuable for many reasons, just not so much for genetic preservation (plants already have a more efficient mechanism for that: seeds) or GMO work.

Seeds can be conserved for very very long times but they are not the same as a specific plant you may wish to maintain, maybe similar but not identical, that requires in-vitro of some sort, or constant maintenance under 24 hours lights for example. All Cannabis seeds made and sold are Heterozygous, none are Homozygous are they?
-SamS

 

[jimbop]

Seeds can be conserved for very very long times but they are not the same as a specific plant you may wish to maintain, maybe similar but not identical, that requires in-vitro of some sort, or constant maintenance under 24 hours lights for example.

There are few advantages to long-term maintenance of plants in vitro over just growing plants in pots. With plants in vitro, one day you come into the lab to find pests have made it into your TC collection, and now everything is contaminated. I've spent entire days rouging shelves after thrips make their way from the greenhouse to the tissue culture lab. Never put your lab within striking distance of your grow room. Never allow people to enter the grow chamber after visiting the grow room. But then that makes it even pricier and more inconvenient, so people do it anyway. One of my buddies runs a major transgenic lab that routinely loses 40% of its plants to contamination, because management refuses to distance the lab from the greenhouses. Air filtration is not the solution to this; only physical barriers will fix it.

The alternative is cryogenic storage, which runs its own risks.

All Cannabis seeds made and sold are Heterozygous, none are Homozygous are they?
-SamS

Zygosity has nothing to do with being in the form of seed vs. plant. It has to do with the dominant or recessive nature of the genes.

 

[Sam_Skunkman]

There are few advantages to long-term maintenance of plants in vitro over just growing plants in pots. With plants in vitro, one day you come into the lab to find pests have made it into your TC collection, and now everything is contaminated.

It is easier to control pathogens and pests in an invitro lab then in a greenhouse filled with plants, I have never had a insect pest in my invitro lab, I made and ran for several years. Insects in a greenhouse can spread viruses to living clones in a greenhouse, I have seen it happen to many clones maintained for over 20 years.


I've spent entire days rouging shelves after thrips make their way from the greenhouse to the tissue culture lab. Never put your lab within striking distance of your grow room. Never allow people to enter the grow chamber after visiting the grow room. But then that makes it even pricier and more inconvenient, so people do it anyway.

Not me. How did they get on the plants in the lab? Were they not in containers that would prevent thrips from being able to touch the invitro plants, cells, meristems, callus?

One of my buddies runs a major transgenic lab that routinely loses 40% of its plants to contamination, because management refuses to distance the lab from the greenhouses. Air filtration is not the solution to this; only physical barriers will fix it.

I have hardly lost anything to contamination even the new xplants that I stick into invitro from my greenhouse I lose less then 10% when I look at them a week or two after they have been disinfected by me and stuck in to invitro conditions. What can I say?

The alternative is cryogenic storage, which runs its own risks.

That was our goal that were were unable to achieve 20 years ago, freeze callus and later regenerate it, but I know people that have done it now. Not so easy though.
-SamS

Zygosity has nothing to do with being in the form of seed vs. plant. It has to do with the dominant or recessive nature of the genes.

I think Zygosity means if the sets of alleles are the same or not, either dominate or recessive?
So do you believe you can find seeds that are exactly identical if the seeds are Heterozygous?
I guess I am saying if you have a special plant, and want to save it, will a seed of the same variety be exactly the same? No, it will not be because each seed is different, they are all a bit different as they are not Homozygous seeds are they? To be identical they need to be Homozygous, I know F1's can seem very Homozygous but they are not and they do have variations, one seed will not be exactly the same as another one. Get it?
-SamS

 

[jimbop]

So do you believe you can find seeds that are exactly identical if the seeds are Heterozygous?

A trait can be heterozygous.

And, yes- two organisms can have identical traits despite being heterozygous for that trait. An example would be AB blood type.

What this has to do with tissue culture is beyond me. It's like comparing rooted clones to seeds. Of course there are differences in genotype between the two progeny.

 

[Sam_Skunkman]

A trait can be heterozygous.

And, yes- two organisms can have identical traits despite being heterozygous for that trait. An example would be AB blood type.

I was talking about Genomes not traits.

What this has to do with tissue culture is beyond me. It's like comparing rooted clones to seeds. Of course there are differences in genotype between the two progeny.

I always thought plants and seeds could be Heterozygous or Homozygous? If I am wrong it would not be the first time, but when I send a leaf or seed to a lab they tell me how Homozygous the genes in the sample are, not a trait, a set or sets of alleles but for the whole genome we tried to create 100% homogenous clones and seeds and came very very close after the S5 generation we had almost two idential sets in the seeds and clones, until fertility issues ended the work with very Homogenous clones and seeds, for all traits.
I still think I am right, but hell I am a self taught guy not a rocket scientist, but that said I have a bit more Cannabis experience then just about anyone. And I am still learning....
-SamS

 

[jimbop]

That's great, Sam.

What does any of this possibly have to do with tissue culture?

 

[Sam_Skunkman]

jimbop:
That's great, Sam.

What does any of this possibly have to do with tissue culture?

SamS:, I was responding to intelliGeneS........,see post below.

Quote:
Originally Posted by Sam_Skunkman
Seeds can be conserved for very very long times but they are not the same as a specific plant you may wish to maintain, maybe similar but not identical, that requires in-vitro of some sort, or constant maintenance under 24 hours lights for example. All Cannabis seeds made and sold are Heterozygous, none are Homozygous are they?
-SamS

You answered:
jimbop:
"Zygosity has nothing to do with being in the form of seed vs. plant. It has to do with the dominant or recessive nature of the genes."

IntelliGeneS had said:

"However, tissue culture would be very valuable for many reasons, just not so much for genetic preservation (plants already have a more efficient mechanism for that: seeds)"

you went on about traits being Homozygous, I said plants and seeds could be Homo or Hertero, as well as traits, that is why it is about invitro. can a whole plant or organism like a seed be Homo or Hetero? I say yes. I think you said just traits can be? One of us is mistaken......
As for tissue culture yes I commented about my hands on experience over 20 years ago.
We grew thousands of meristems, and callus and single cell suspensions, I had the lab built, I did the lab work, we had refrigerated racks, frozen racks, all with lights at whatever hours we wanted. We made our own mediums for the petri and tubes we used, we also had it made by professionals, 25 years ago. Tissue culture is pretty easy if you can follow instructions well. I used "Plants from Test Tubes: An Introduction to Micropropagation – Illustrated, 1983 there is a new 4th edition out now, great book by Lydiane Kyte (Author), John Kleyn (Author) I just did what they said to do, almost zero problems until shooting callus and freezing callus , cells , or meristems, not much luck with that, no one could do it well back then with Cannabis. Every one can now.
I am a amateur who tried hard and had great success, just like many folks on this site could be if they tried. I still remember the first little clone an inch or two tall flowered completely in vitro in agar gel it was very cool, very small.... My real dream was to use it as a library, frozen, thousands in a very small area, one of several, ultimate back ups.
I presume very soon this will be a reality, someone will do it, freeze Callus or meristems or cells, and regenerate them. Chimera is pretty close to having a methodology that works.
-SamS

 

[GlandualFever]

My real dream was to use it as a library, frozen, thousands in a very small area, one of several, ultimate back up.
-SamS

Why did this not become a reality Sam? I mean you have one of if not THE most comprehensive library of cannabis genetics in seed form and you are still collecting for your DNA project..

 

[Sam_Skunkman]

I do not have a lab....... When I did you could not do this.
Collecting DNA for sequencing is much easier then freezing the same seeds/clones as a library as living plants to keep them alive. I am sure others will.... It can be done now...
-SamS

 

[Daub Marley]

What would be very interesting to me is if anyone has had success generating haploid plantlets or callus of Cannabis via anther/ovum culture or similar methods. This would be a step forward in tool development to help start to resolve the current "pheno-hunt" business methodology in Cannabis breeding which results in a highly inconsistent product being widely sold. DH breeding might be a powerful strategy to pursue.

I just happened to learn about anther/ovum culture yesterday and I am shocked this hasn't been popularized yet. Am I missing something?

 

[purple_man]

high fambz!

ovule n anther cultures are being done, double haploids are a nice tool for doing fast paced "inbreeding" or for finding interesting expressions when created from a true hybrid. it's used intensively in wheat breeding, ...

blessss

 

[burnone710]

Can a culture be taken from a plant that has been flowered out already during harvest or can you re-veg and take a culture? Will this save the genetics without losing the vigor and potency that you would normally after re-veg ? I have an amazing cut I misjudged and killed the mother too early, still have it flower almost a week from harvest...tissue culture kit arriving around harvest time. Really wanna get this sucker back!!