Tissue Culture/Micro Propagation Grow from Scratch

Wolverine97 · Mar 15, 2011

haole420 said:
3 weeks and no roots? not sure what benefits are to be had with tissue culture over cloning.

kudos to canndo for actually doing this experiment, though.

if you used tiny tissue samples (hole punched leaf) and ramped this up to commercial production scale (i.e., starting a new batch of thousands of tissue samples every day), then it might make sense. i think that's why they use tissue culture for large-scale crops like bamboo, strawberries, asparagus, etc.

unless you're growing tens of thousands of square feet of weed, i'm not sure tissue culture is worth all the trouble when the same can be accomplished by cloning. plus with cloning, you can take bigger clippings that'll put you at least a month ahead of this kind of propagation in terms of growth.

keep it going though: i want to see how they end up!

You have to switch to a different hormone to induce rooting. Tissue culture is different from normal cloning since you're dealing with such tiny pieces of plant matter, you have to first induce shooting, then rooting, at least that's my basic understanding of it.

canndo · Mar 15, 2011

I am not sure what clonex has in it as an auxin. Nothing has roots because I don't WANT roots yet. The moment we see roots on a shoot it stops multiplying, fills out and begins more normal growth. The experiment entails establishing an explant, growing shoots large enough to take them and place them into a new batch of medium and then when they get large enough, placing those in rooting medium (and finally, hardening them off, growing them to a decent size - 1 foot for this experiment - and likely flowering) It is the hardening off, the taking them from a controlled and almost womblike environment into a light, dry harsh world that has me concerned.

Haole, I have never been able to get anything but callus to grow from a leaf cutting. If you look closely at some of the pictures you may see that where ever a leaf touches the medium, they turn sort of brown and get flabby. This is all I have ever gotten. I perfected a pretty good callus growing medium early on but I have yet to get differentiated growth from that callus mass.

But here is the thing. As I said, I have gotten over 50 "clones" from only a handful of explants. One could easily call each of these "clones" mother plants - each giving me an average of 3 "cuttings". I think another cytokinin tweak could give me more - the balance is that the more cytokinin the leggier the shoots, not such a good thing when the plants are so small.

In effect I have on one shelf and under a single fluro, 50 different sources of genetics which will each give me at least 3 cuttings every three weeks. That alone is 150 "clones". And that is if I only place one explant in each vessel. I don't water the things, I don't have to do anything with them until "transplant" and that takes a little more than an hour. Now I am not sure with this plant but I know that in vitro plants generally have extraordinary vigor. I do not believe such a technique will ever replace someone taking some cuttings from his coupla moms hidden under a CFL in the basement. I do think that it has a place in transporting, preserving and mass production. I can keep backup genetics in a different location, I've slowed growth and kept them alive in a refrigerator.

Advanced techniques include embryogenisis and encapsulation which would give us the ability to have EXACTLY the genetics we want from artificial seed. That is still a ways off but the techniques are sound and you have to start somewhere. One of the reasons I started doing this is because I was sick of "weeding" through different phenos trying to find the phenotype everyone else was raving about.

Wolverine97 · Mar 15, 2011

canndo said:
I am not sure what clonex has in it as an auxin. Nothing has roots because I don't WANT roots yet. The moment we see roots on a shoot it stops multiplying, fills out and begins more normal growth. The experiment entails establishing an explant, growing shoots large enough to take them and place them into a new batch of medium and then when they get large enough, placing those in rooting medium (and finally, hardening them off, growing them to a decent size - 1 foot for this experiment - and likely flowering) It is the hardening off, the taking them from a controlled and almost womblike environment into a light, dry harsh world that has me concerned.

Haole, I have never been able to get anything but callus to grow from a leaf cutting. If you look closely at some of the pictures you may see that where ever a leaf touches the medium, they turn sort of brown and get flabby. This is all I have ever gotten. I perfected a pretty good callus growing medium early on but I have yet to get differentiated growth from that callus mass.

But here is the thing. As I said, I have gotten over 50 "clones" from only a handful of explants. One could easily call each of these "clones" mother plants - each giving me an average of 3 "cuttings". I think another cytokinin tweak could give me more - the balance is that the more cytokinin the leggier the shoots, not such a good thing when the plants are so small.

In effect I have on one shelf and under a single fluro, 50 different sources of genetics which will each give me at least 3 cuttings every three weeks. That alone is 150 "clones". And that is if I only place one explant in each vessel. I don't water the things, I don't have to do anything with them until "transplant" and that takes a little more than an hour. Now I am not sure with this plant but I know that in vitro plants generally have extraordinary vigor. I do not believe such a technique will ever replace someone taking some cuttings from his coupla moms hidden under a CFL in the basement. I do think that it has a place in transporting, preserving and mass production. I can keep backup genetics in a different location, I've slowed growth and kept them alive in a refrigerator.

Advanced techniques include embryogenisis and encapsulation which would give us the ability to have EXACTLY the genetics we want from artificial seed. That is still a ways off but the techniques are sound and you have to start somewhere. One of the reasons I started doing this is because I was sick of "weeding" through different phenos trying to find the phenotype everyone else was raving about.

Yeah, I totally understand that you haven't switched hormones to induce roots yet. I was just saying in general, I want to see this process completed.

Thanks for sharing the info, I'll hang up and listen.

canndo · Mar 15, 2011

Woverine, I would dearly love to just send these plantlets into root in my next pass but it would really prove very little to the folks who have made growing this plant their hobby or vocation. After all, I will wager that almost everyone here has taken a cutting and set it to root in one way or another. That is pretty much what I would be doing. Thank you very much for your interest, I'm glad to have it.

Wolverine97 · Mar 15, 2011

canndo said:
Woverine, I would dearly love to just send these plantlets into root in my next pass but it would really prove very little to the folks who have made growing this plant their hobby or vocation. After all, I will wager that almost everyone here has taken a cutting and set it to root in one way or another. That is pretty much what I would be doing. Thank you very much for your interest, I'm glad to have it.

Understand. It's just my selfish side coming out, I've never seen tissue culture documented start to finish. I guess the real process is in the perpetuation though, and I'd really like to see how long you can continue pulling shoots from a host also. I'm just really curious about this whole thing. I'm a pretty detail oriented guy, and constructing a semi-clean, positive pressure box wouldn't be too much trouble for me.

If you keep this thing going, I'm sure I'll have questions for you. This will be the first time I've actually asked questions on here, I generally try to help the guys having "nute problems" or what have you, but you have my full attention.

Thanks again for posting this, really.

Edit: If rooting the plantlets isn't any different than rooting a normal cone, then do you really have to sweat the exact ratio of hormones? Or is it just because they're so fragile?

canndo · Mar 15, 2011

The problem with roots is that they themselves are very fragile. If I take a rooted plantlet out and put it in soil, the roots might shear and then there would have been no point. The goal then is to have a protocol that will give me the hardest possible roots and in the softest possible media. The roots will have to actually function. You see, this plant is almost completely on life support - it does not manufacture it's own energy, photosynthisis is almost at a standstill, stomata do not function as far as opening and closing, there is a fundamental difference in the physical structure of the plant's surface. Roots may be the same way so we have to stimulate them to be as strong as possible. Mind you I am inventing none of this in general, it is only that there are very few studies on this particular plant.

BTW I am on generation 5, I believe I've posted pictures of that. I can't go much further until I figure a way to vent the vessels.

stupidclown · Mar 15, 2011

can you use tyvex?(spelling?) its the fabric usps priority envelopes are made of, you can use it in mushroom jars it lets in a small amount or air but filters out everything else.

Wolverine97 · Mar 15, 2011

stupidclown said:
can you use tyvex?(spelling?) its the fabric usps priority envelopes are made of, you can use it in mushroom jars it lets in a small amount or air but filters out everything else.

Actually, Tyvek lets moisture vapor and air escape, but doesn't allow it to infiltrate. It works sort of like a one way valve.

Wolverine97 · Mar 15, 2011

canndo said:
The problem with roots is that they themselves are very fragile. If I take a rooted plantlet out and put it in soil, the roots might shear and then there would have been no point. The goal then is to have a protocol that will give me the hardest possible roots and in the softest possible media. The roots will have to actually function. You see, this plant is almost completely on life support - it does not manufacture it's own energy, photosynthisis is almost at a standstill, stomata do not function as far as opening and closing, there is a fundamental difference in the physical structure of the plant's surface. Roots may be the same way so we have to stimulate them to be as strong as possible. Mind you I am inventing none of this in general, it is only that there are very few studies on this particular plant.

BTW I am on generation 5, I believe I've posted pictures of that. I can't go much further until I figure a way to vent the vessels.

But you have to be able to move them to soil at some point, right?

canndo · Mar 15, 2011

Otherwise, the excercise would be interesting but rather pointless.

canndo · Mar 15, 2011

Tyvek, I am trying to figure a way to attach it to the magenta caps and still withstand autoclave. What I have found in starting this project is that there really is very little for the common person (not the posted project but the process of doing this as a whole). The vessels are hard to find, hormones are difficult, shipping is nuts for some of this stuff. I find that there is always some adaptation - like using test tubes - they suck. Like using non-absorbant cotton instead of proper filtration - it sucks worse. Try pricing baby food jars forgodsake. I've gone through almost a dozen different hormones (or hormone like substances) and I've had to buy huge amounts of them only to use a few tenths of a miligram.

canndo · Mar 16, 2011

During a recent media run I tried using some larger tubs and I discovered I had made some jars I didn't account for. A week ago I exposed all of them to the air in my kitchen for 1 minute for the jars and 2 minutes for the tubs. Now I wouldn't recommend doing this in real conditions but it goes to show that you can do transfers in a room with a standard sort of room hepa cleaning filtration system you can buy at Home Depot for 80 bucks if you don't go swishing the air around and you use biocides in your media. I am going to do a big media run today.

canndo · Mar 16, 2011

No contamination in any of the jars or tubs.

canndo · Mar 17, 2011

Ok, all trimmed and transplanted. I started with 12 jars, about 15 explants. I lost 6 jars total for one reason or another, this is a higher loss rate than I am used to but it doesn't matter that much in the establishment phase so long as I get a few good ones established. I now have 10 jars of established explants, each of these is a shoot that was grown in vitro. I waited too long to do the transfer and some of the shoots were wilting or I would have had more. Seems that the stiffer medium cuts my time so I don't have much of a luxury to wait a few extra days. I throttled the gelzan back down to only 5 percent more than before but it looks like they will all stand up.

I am thinking of posting some more experiments that I am doing today - namely germinating seeds in vitro. Finally, and I will try to get a picture - I opened an establishment jar that was 3 weeks old when I opened the fresh jars of media. It is now covered with contamination. Seems the biocide has an active life of about three weeks give or take. Just something to note.

If all goes well, some if not all of these plantlets will be grown out for a few weeks and then transplaned into rooting media. I think I will just reverse the Cyt/aux ratios.

canndo · Mar 17, 2011

I got a carmaliscous branch in today, it has seriously thicker stems and I am considering putting those pics in with this rather than starting another journal. Same with the seed. I don't know how it works here. There are so many microcultivation threads I'd hate to start yet another over this, someone let me know which would be the more appropriate if you will?

Wolverine97 · Mar 17, 2011

canndo said:
I got a carmaliscous branch in today, it has seriously thicker stems and I am considering putting those pics in with this rather than starting another journal. Same with the seed. I don't know how it works here. There are so many microcultivation threads I'd hate to start yet another over this, someone let me know which would be the more appropriate if you will?

Definitely keep it in this thread, the more condensed the better. Keep it coming man, good stuff here.

canndo · Mar 17, 2011

fine. 1 5 second Isopropyl wash, regular water rinse. 1 more 5 second isopropyl wash, 1 more rinse. 20 minutes 5 percent bleach. 15 10 percent and 5 15 percent. The 15 percent is in the ultrasonic right now. A couple of sterile water rinses. Then I will hit it with 1 minute 91 percent isopropyl and do 8 washes with sterile water. No point in pictures, it looks the same as the last time, just a heartier wash because the stems look like they can take it. If you see bleaching of the ends - you are done, move on, it will still likely work.

canndo · Mar 17, 2011

They are all in, some of the biggest explants I've yet tried. No pictures tonight. I still have the seeds to finish.

canndo · Mar 17, 2011

When I was young I wound up throwing quite literaly pounds of seeds. Now I had trouble getting 14 without paying for them. They are mostly seeds that I found in flawed buds and such. They are all now in vitro.

Less sucrose, different ph (6.

I don't know how the seed hulls withstood the beating I gave them but I had nothing but trouble with sterilizing the last batch I did.

canndo · Mar 18, 2011

More stuff:

I wondered if rather than cook up a new batch of media every time the plants began to grow slower or droop if I could just move the plants a bit to one side or another. The plantlets don't grow roots (yet) so the only nutrient they get must be adjacent to the stem. Once the sugar in proximity to the stem is depleated the plant's growth will slow. I don't know if the PH (rather critical in this process) is altered by any exudate from the stem - it might, but even so, even as the sugar likely does not migrate through the gell, exudates probably do not either. I am finding that the biocide expires and I suspect that the hormones have a limited working life being exposed to moisture, light and heat. But the question still remains. At some time in the future I will try just such a move.

Tissue Culture/Micro Propagation Grow from Scratch

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