Tissue Culture/Micro Propagation Grow from Scratch

canndo

Well-Known Member
Perhaps Wolverine, this process will enable us to send exact genetics through the mail. Wouldn't that be wonderful. I did a complete scan of my starts this morning. I took another loss - it looks like bacteria. If I don't put an end to my other endeavor it will have an ever growing effect on this one. I consider the micropropagation much more important.



Ok, so the seeds seem to have moved, just barely so I can't call the experiment failed.
DSCF1158.JPG
DSCF1157.JPGThese are bottom shots so that you can see the bacterial infection, they are a bit blurry but you can see a sort of cream/yellow smear around the base of the plant. The biocide can get overwhelmed and when it does this is the result. Of course I have yet to salvage a plantlet from a contaminated jar. I believe I could do it if I use the biocide straight on the plantlet but the plantlets aren't worth the cost to me.

.DSCF1153.JPGDSCF1154.JPGNow look at the particulars of this contamination. The hairs are briliant white, they grow roughly parallel to each other and they have not yet exhibited spores or coloration. This may be a genuine first - one psychonautical organism eating another.

DSCF1159.JPGas I said, bacterial contamination. I don't want anyone to be discouraged - this stuff happens and it is easily controled. Once you get started, the worst thing about contamination is the bother of cleaning the jars. So long as you catch it early, you don't even have to re-sterilize the contaminated units. It is just dissapointing because as I said, I like all of them and I hate to see any wasted. We should be getting back to normal in just a few more days when I halt the other project.


DSCF1155.JPGDSCF1156.JPG This is my mistake. I chemicaly "cooked" this plantlet with alcohol, bleach, more bleach, more alcohol and I had it in the unltrasonic cleaner for a long time. It just never made it - I really wanted to see this one take off too.

DSCF1160.JPG
This is one from that same branch - note the thick stem, that worked. I can't put all pictures of failure on here, I have to put a little sucess on this post. This plantlet was taken last week and it shows the characteristic first shoot growth, remember that I cannot put cuttings in that actually have leaves. The leaves are all but impossible to disinfect and then because the tissue is so thin, the leaves are killed. Contrary to popular belief, working with leaves of C. Sativa currently gets us nowhere. You can create callus from leaves but organogenisis - making undifferentiated cells into shoots is exceedingly difficult. Furthermore, callus tends to impart uncertain genetics to the plants that are spawned from it. Part of what I would call a "break through" is the acceptance that there needs to be no callus phase in the proceedure. Callus works for some plants but until someone comes up with a better way to shoot from callus, it doesn't work very well here.
 

canndo

Well-Known Member
Thanks, I believe I do. Got a heating mat and some new solid media for the seeds. I will rig up something new come Tuesday - more seeds, more cuttings. Back to work - thanks for your input woverine and thanks to everyone else reading this.
 
First off, canndo, this thread is a pleasure to learn from, truly a "neutral pipe": all the information with no noise. An outstanding journal about an unique topic.

I hope it's ok to post this link because you had said you hadn't seen much on cannabis tissue culture:

http://www.pakbs.org/pjbot/PDFs/41(2)/PJB41(2)603.pdf

I'm just a closet farmer, so this technique is mostly a curiosity for me, but do I understand that one of your goals is to produce seeds in vitro? I'd love to put some mothers in a cool, dark place, just in case.

Sub'd.
 

canndo

Well-Known Member
Rollman, I would like to have created artificial seeds but that will take another breakthrough. My goal is to be able to micropropagate in a non-sterile environment. I had wanted to be able to create artificial seeds but barring a breakthrough in organogenisis I don't think that particular shortcut to being a seed seller is viable.

Thanks for the link to that Chinese study. I've seen it before, it is a very valuable start.
They have some of the clues but I have found that we can do better than the TDZ and NAA alone.

When I said I hadn't seen much, what I meant was that I hadn't seen much on forums that went anywhere.


And gee - thanks VERY much for the complements. I would love for you to be able to put mothers in a cool place undisturbed as well.
 

USMC

Member
Hey Cando,

Very informative thread. I remember in one of my biotech classes we attempted culturing, obvioulsy not with MJ. We all used leaf material and only a few groups got any viable cultures. Im going to attempt this in a month or two after I get a written protocol together. Is there any other reasonably priced lab equipment that you would like to have had? Perhaps a UV light box for sterilization?
Are there any premade media's that you can purchase from Sigma, Stem Cell, or some other distributer that you may have used?

Thanks again for your posts.

Cheers
 

canndo

Well-Known Member
No, nothing else. I was surprised at how adaptable it all is to anyone who is at all ingenious.
 

canndo

Well-Known Member
DSCF1172.JPGDSCF1173.JPGDSCF1175.JPGThese are pictures of the latest cuttings, it shows that indeed, the larger stems seem to grow more quickly and more solidly, the center one is quite young. Note the last one, it was burned very badly but still mangaged a comeback. I am used to thinner cuttings but it seems that the thicker ones need only a bit more cleaning. DSCF1174.JPG I am doing this again today with two unknown varieties. Sorry that these pictures aren't of some beautiful buds all sticky and glistening.

DSCF1163.JPGDSCF1169.JPGDSCF1168.JPGDSCF1166.JPGDSCF1164.JPGDSCF1171.JPGDSCF1170.JPG I figured I'd fish some out of the jars so that you could see them better. These are all large enough to either take new cuttings from or simply root. They were in groups of three where one of them was infected so I figured I'd show them off before tossing them. I've tweaked the formula yet again, bumping up the GA. As you can see, they are still rather compact and tend to like sending up new shoots - my average is still three though.

DSCF1177.JPG DSCF1176.JPGNow these are two different views of our original Lemon Skunk. They look pretty healthy for going over a month and recall that this was from a single micro-cutting that was divided into three last week. The others look fine but they don't quite look as good as these three. This process does indeed give me more (although not yet rooted) stock than I need in short order. Note the start of more shoots in there, especially the second one and to the right.
 

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canndo

Well-Known Member
Oh. And the seeds? No go. I can't figure out how the Chinese managed to do it but I can't get seeds to grow in sucrose. It is not a heart or deal breaker, I just wanted to get them to grow from the very beginning in gel. I am doing a batch right now for some unknown plants that were given to me yesterday. Even though I've been doing this for what seems like a very long time, I still enjoy the work, seeing them come up from seemingly dead little sticks and of course I need a lot of stock for future experiments.
 

Wolverine97

Well-Known Member
View attachment 1522264View attachment 1522260View attachment 1522259These are pictures of the latest cuttings, it shows that indeed, the larger stems seem to grow more quickly and more solidly, the center one is quite young. Note the last one, it was burned very badly but still mangaged a comeback. I am used to thinner cuttings but it seems that the thicker ones need only a bit more cleaning. View attachment 1522257 I am doing this again today with two unknown varieties. Sorry that these pictures aren't of some beautiful buds all sticky and glistening.

View attachment 1522265View attachment 1522262View attachment 1522255View attachment 1522253View attachment 1522252View attachment 1522263View attachment 1522254 I figured I'd fish some out of the jars so that you could see them better. These are all large enough to either take new cuttings from or simply root. They were in groups of three where one of them was infected so I figured I'd show them off before tossing them. I've tweaked the formula yet again, bumping up the GA. As you can see, they are still rather compact and tend to like sending up new shoots - my average is still three though.

View attachment 1522258 View attachment 1522261Now these are two different views of our original Lemon Skunk. They look pretty healthy for going over a month and recall that this was from a single micro-cutting that was divided into three last week. The others look fine but they don't quite look as good as these three. This process does indeed give me more (although not yet rooted) stock than I need in short order. Note the start of more shoots in there, especially the second one and to the right.
Looking good canndo, it looks as if you're getting the process refined a bit. The shoots are looking healthy, much better start than the first set you posted. Keep 'em coming!
 

Wolverine97

Well-Known Member
Oh. And the seeds? No go. I can't figure out how the Chinese managed to do it but I can't get seeds to grow in sucrose. It is not a heart or deal breaker, I just wanted to get them to grow from the very beginning in gel. I am doing a batch right now for some unknown plants that were given to me yesterday. Even though I've been doing this for what seems like a very long time, I still enjoy the work, seeing them come up from seemingly dead little sticks and of course I need a lot of stock for future experiments.
Is there any way to maybe make the medium form a bunch of micro air bubbles? I wonder if that would make a difference, I've got to think that oxygen is the limiting factor there...
 

canndo

Well-Known Member
If I use a completely air tight system and shake it before it congeals I suppose it would work - there is a critical period when the gel sets. I know about the o2 - Sucrose has oxygen in it but I don't know if the plant uses it or not and it sure isn't free oxygen.
 

Wolverine97

Well-Known Member
If I use a completely air tight system and shake it before it congeals I suppose it would work - there is a critical period when the gel sets. I know about the o2 - Sucrose has oxygen in it but I don't know if the plant uses it or not and it sure isn't free oxygen.
I don't know what the technical name is, but do you have a shaker plate/thing? If you manually shook it to create air pockets, and then vibrated it to get the bubble size and consistency a bit smaller maybe it could work. I have no idea, obviously, but maybe it's worth a shot.
 

canndo

Well-Known Member
It is worth a try, I have a shaker, or rather a swirling mechanism - next pass as he current one is already hard.
 

canndo

Well-Known Member
Today.

I put off doing the cuttings yesterday because I ruined my media - remember never to pour gell compound into warm water. I thought I could even it out with enough stirring and then cooking but a lot of the jars failed to solidify. I am not as worried about this as I am the lack of consistancy. I have learned from doing this and other things that so long as you take notes on everything you do, it is a good idea to occasionaly screw things up - that is how discoveries are made. I accidentaly put Dicamba in solution, and then, forgetting, I did it again - the double dose was just what was needed to promote vigorous callus. If I hadn't made notes, I wouldn't have known. Too bad that was a dead end.

Anyway, I am quite certain now that stem girth promotes extraordinary growth if the GA is up there. This is the largest and fastest growth I have ever seen in all my trials. I will attempt to duplicate this one again but use 10 seconds alcohol, 10 minutes 5 percent bleach, 10 minutes 10 percent bleach and then 20 seconds alcohol. Shit, I forgot to sterilize my water -
 

canndo

Well-Known Member
That's better, but now I am going to have to wait for the jars to cool. Nothing more to do just yet so I figure I should begin to pretend I am an expert and teach, maybe someone else will follow in my aparently pioneering footsteps.

What do you really need to do this?

1. A pressure cooker - preferabley one that will hold everything at one time but I think you could get away with a what? 5 quart?
2. Jars - lots and lots and lots of jars, the size doesn't matter but frankly I find babyfood jars to work for most things. Once you get some going those tubs I am using are pretty handy and so far as I can tell so far, the plants like the extra space (more air - I am certain of it). I am thinking of using the pint small mouth canning jars as well. I figure I can put together some clear plastic disks for the lids - I can put as few as one and as many as half a dozen in each one. The point you are getting to is the use of as little media as possible - half an inch or so in the bottom.
3. Along with the jars are some sort of lid. Air tight is one way, although I am finding this to be less than perfect. However, even with the biocide, you can't just have a hole in the lid without some sort of filter
4. light - any growlux will work, I've not experimended with light too much because I figure if I have green plants the light is good enough.
5. Instruments - a couple of long tweezers, a couple good scalpels - disposable are ok but... you go through a lot of them. I found a disposable that can be autoclaved - the best of both worlds - have a new one handy and sterilize some of the ones you have already used.
6. A turkey baster, don't skimp on this one - really, spend the 5 bucks or so - you will thank me.
7. A screen disc that fits on a small mouth canning jar - punching holes in the conventional disk just doesn't work right
8. A good PH meter - really, a good one, it would be better if you can get one that goes to 100ths rather than just 10ths. I have found that ph is very very critical and being off a 10th will screw the whole thing up.
9. PH up - yep, but if you have the ph meter then you probably have the KOH don't you.
10. Mixing or agitation system. Now that is two things actually. There must be a way to clean your plants, in order to do so you will need to place them in a low surface tension liquid with anti microbial and anti fungicidal properties and agitate that solution - this is critical. You will also probably want something to help you mix the media but this is not critical, as shaking will do.
11. 91 percent isopropal alcohol
12. Bleach
13. An HEPA room filtration system - one of the cheaper ones will do nicely - a few hundred dollars.
14. The various media -

I don't think I left anything out - everything else is an embelishment, something to make the process simpler or more reliable.
 

Illumination

New Member
Mescaline, psylocybin and that was all. Mescaline was very very rare and mushrooms handn't even been invented yet.
How can someone who stated this be doing what is happening in this thread...I am utterly and totally overwhelmed with confusion

Namaste':leaf:
 

canndo

Well-Known Member
Illumination? ever hear of exaggeration for effect? how about utter silliness? The specifics are this - when I was young the methods for growing mushrooms in one's kitchen had not been invented, the turning point came with Oss and Orick's tiny book sometime in the late 70's. It was a pivotal event in the evolution of mind expansion. I hope to be instrumental in another such pivotal event. Be not afraid of confusion for it is the distant beginning of clarity. And kindly watch this journal if you would.
 
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