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Tissue Cultured Marijuana ? You Bet !


trichcycler

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I saw that in the book but I've never seen it personally, yet. Mine have all been a light green and were very firm when touched. what area do you cut your samples from?

 

 

also, tell me those eggs and mites were dead when you discovered them?  I don't suspect they will survive a sterilization?   with that I wonder how they could cause these curled leaves in vitro?   I see curled leaves in vitro also occasionally, but not in veg/flower. When I maxed some hormones, salts etc the plantlets would show the same issue for a bit, but most of my samples were smaller than a seed to begin with. By the time they chute/root their little bonsai tanks with no turning back. When I've taken samples the size of yours, to store, that's when I saw some curling leaves. I cut some salts and the issue went away.

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I saw that in the book but I've never seen it personally, yet. Mine have all been a light green and were very firm when touched. what area do you cut your samples from?

 

 

also, tell me those eggs and mites were dead when you discovered them?  I don't suspect they will survive a sterilization?   with that I wonder how they could cause these curled leaves in vitro?   I see curled leaves in vitro also occasionally, but not in veg/flower. When I maxed some hormones, salts etc the plantlets would show the same issue for a bit, but most of my samples were smaller than a seed to begin with. By the time they chute/root their little bonsai tanks with no turning back. When I've taken samples the size of yours, to store, that's when I saw some curling leaves. I cut some salts and the issue went away.

Most of the callus I've got was from leaf tissue. It was the first TC I tried doing last Sept.

 

Regarding the russet mites, I guess I need to explain a bit better. I have a couple small moms that I was getting cuttings from for experimenting...someone had given them to me. I think one of them brought the mites in and infected the other one, as well as a couple more plants that came and went. The photos I posted showed the leaves of one of the moms. The photo of the culture jar was of one of my cultures that I had thought was infected, too. I think I pushed the panic button on thinking the culture jars were infected, but they are not. The mites I saw were on the live mom and definitely alive. I've come to believe the curling of the leaves in vitro is from vitrification and I'm going to approach a solution from that angle. I'm going to use a different cytokinin for starters and see how that works; perhaps cut back on the salts. Sometimes less is more, as they say. Regardless of any issues with mites, or not, I'm wrapping my jars from now on and will try to wean myself off of PPM.

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wow Your first culture!!! Congratulations!

 

Leafs are where I began too. I choose  a new apical stem now for the fastest results here. I have cultured anthers too, and calyx, roots are especially fun.

 

PPM saved my hobby for years, good luck working without it. is possible, but was not for me for awhile. Using it would afford me higher success rates for thought.

 

My first hundred were simple fungi farms lol, then once in awhile success would happen, and I mean just positive growth callus. It was decades until I could get a chute, and another till I got roots, then roots and chutes followed, then years into the hardening off stage, then finally success, sort of. Then came the weirdo's my work created, altered genetics, twisting dying plants, failures and so on. Recall I had no internet or library tissue culture information available to me except tutelage. I was able to work carrots, tobacco, venus fly traps, bananas etc  at every stage, transplanting including,  but cannabis proved more difficult for me. Even after sterile tech is acceptable the cannabis plant seems different than trees or tomatoes or tobacco for me in culture. a lot of those are near interchangeable recipes for support, but not mj sadly. Some of those mentioned are available to purchase for practice. The experience was good for me and a cheap way to jumpstart the education imo.

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wow Your first culture!!! Congratulations!

 

Leafs are where I began too. I choose  a new apical stem now for the fastest results here. I have cultured anthers too, and calyx, roots are especially fun.

 

PPM saved my hobby for years, good luck working without it. is possible, but was not for me for awhile. Using it would afford me higher success rates for thought.

 

My first hundred were simple fungi farms lol, then once in awhile success would happen, and I mean just positive growth callus. It was decades until I could get a chute, and another till I got roots, then roots and chutes followed, then years into the hardening off stage, then finally success, sort of. Then came the weirdo's my work created, altered genetics, twisting dying plants, failures and so on. Recall I had no internet or library tissue culture information available to me except tutelage. I was able to work carrots, tobacco, venus fly traps, bananas etc  at every stage, transplanting including,  but cannabis proved more difficult for me. Even after sterile tech is acceptable the cannabis plant seems different than trees or tomatoes or tobacco for me in culture. a lot of those are near interchangeable recipes for support, but not mj sadly. Some of those mentioned are available to purchase for practice. The experience was good for me and a cheap way to jumpstart the education imo.

 

Actually I didn't know what I was doing when I did those first cultures. I just took some leaf material and stuck it in a dish with the stage 1 media, then to the stage 2 media from TriQ systems. I will say that their s-2 grows callus very well. However, due to different issues with their media I didn't continue using it. Realized it was much less expensive to make my own (which I do for about 3 bucks a liter). So I had to re-learn chemistry that I slept though in HS when I was stoned, lol. It's been fun, getting the "aha moments" when one connects the dots. Having a biochemist as my next door neighbor has helped a lot, too! Haven't really tried growing much callus lately, although I do have a good recipe for it. One study I read found that the petioles work best for callus and will be trying that in the near future after I get my moms cleaned up. I've noticed callus growing at the base of nodes that I put into culture, but have thought that was a detrimental thing. After reading about carrot TC, perhaps it is not.

 

Here's my situation, presently, and it's what got me into TC in the first place. A friend, who's a grower approached me because he knew I did mushroom culture. (I know I'm repeating part of this story, but bear with me). He had two small moms that had started flowering and wanted to save the genetics. The Z7 ant the QTZ. Being full of trichomes, they were also full of contamination. I took the part that wrorked the best initially...the leaves. Later I was able to coax the plant back to veg state and got some good nodes to culture. Still have some of those stable in culture but fear I will lose them. Some of them look healthy enough to clip some petioles so I can make callus and get it to shoot and root at a later time when I'm a bit more accomplished at his art from.

 

Since then I've been approached by several growers for clean starts. If I could produce them, I could have orders for several thousand starts. Of course, I don't have the technique or space for such an operation. However, the guy who initially approached me is willing to build a lab for such an operation, as well as one other person, too. I'd like this to be my "second career" as I approach retirement age. I'm a bit shocked that it took so long for you to arrive at a suitable protocol; it's also a bit discouraging. But, I will keep at it, nonetheless. I understand I have many more resources at my disposal than you had for many of your years and I have a lot of determination, as well as humility because I don't have the background many others in this business have. BTW, it won't be anytime soon that I go off of PPM, just something I think about. I want to simplify things as much as possible, and perhaps PPM allows that, for now. I really appreciate having someone to talk with about this stuff...thanks!

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Found some roots growing on this plantlet today. It was sort of stalled in it's rooting medium but took off a couple days ago, growing very well and today there is at least one root showing...two weeks to the day. Hope the rest follow suit. It seems like every step of this process is a challenge. To complicate matters it also seems like every variety responds differently to culture conditions. At any rate, the next challenge is hardening off, from which I understand, can be quite the art-form.post-39662-0-04830900-1455402630_thumb.jpgpost-39662-0-86244700-1455402638_thumb.jpg

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Organogenesis from callus tissue of a high CBD medicinal plant. Funny thing is, this is happening on a formula for expanding callus, not from a shoot formula, containing both an auxin and cytokinin. Going to try some formulas containing only an auxin and only a cytokinin. Will I get only roots, or only shoots? I don't know, but it will be fun trying! 

 

post-39662-0-97718900-1455668957_thumb.jpeg

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I've got about 20 jars with this strain of callus in them, this one is the most advanced but many others are starting to get little green dots in them, so I've got plenty to work with. This particular one also looks as if some rooting may be in the works. The formula, taken from a study, was supposed to be for callus induction and growth, but the same cytokinin/auxin ratio and ingredients are also used (in another study) for shoot induction from callus as well as a multiplication medium. 

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  • 2 weeks later...

The leaves shown in some of your pics.......are those cultured emerging chutes or did you use cuttings for the explant sample? 

 

I use cuttings this way to store them in stasis. Sometimes even when not ordered to do so, the cuts will begin to multiply and even root.

I leave them alone mostly for the awe factor.   They store in test tubes for along time without fuss. My longest without change up is 2 years.

They're fine without light for a couple days but then they begin to show distress. (shipping concern). I've stored one in a dark envelop for weeks

using only a  small led white light, the type that snaps on top of a 9 volt battery. (like this one http://www.amazon.com/Anself-Fashion-Blocklite-Flashlight-Camping/dp/B00MGMRYWC/ref=pd_sim_468_3/191-8034199-1085944?ie=UTF8&dpID=41RXOPnNY0L&dpSrc=sims&preST=_AC_UL160_SR160%2C160_&refRID=0EHJKAMFDAY8A63309TY)

My battery recently lasted around one week on the low setting before needing replacement. One week is acceptable to me. The sample was healthy and lives in dirt today. When its finally legal this will be a viable genetic exchange route for someone.

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The leaves shown in some of your pics.......are those cultured emerging chutes or did you use cuttings for the explant sample? 

 

I use cuttings this way to store them in stasis. Sometimes even when not ordered to do so, the cuts will begin to multiply and even root.

I leave them alone mostly for the awe factor.   They store in test tubes for along time without fuss. My longest without change up is 2 years.

They're fine without light for a couple days but then they begin to show distress. (shipping concern). I've stored one in a dark envelop for weeks

using only a  small led white light, the type that snaps on top of a 9 volt battery. (like this one http://www.amazon.com/Anself-Fashion-Blocklite-Flashlight-Camping/dp/B00MGMRYWC/ref=pd_sim_468_3/191-8034199-1085944?ie=UTF8&dpID=41RXOPnNY0L&dpSrc=sims&preST=_AC_UL160_SR160%2C160_&refRID=0EHJKAMFDAY8A63309TY)

My battery recently lasted around one week on the low setting before needing replacement. One week is acceptable to me. The sample was healthy and lives in dirt today. When its finally legal this will be a viable genetic exchange route for someone.

 

The leaves coming out of the callus are regenerated from the callus, not from a cutting. The original cutting was just a small section of leaf that grew into the callus from which the new leaves came. They are very slow growing at this point and it's the only piece of callus that has grown leaves. The other calli just sit there, expanding, some green, some yellow. 

 

So, the idea with the battery/LED in the envelope would be for future shipping purposes? 

 

How long does it generally take for enough roots to form to move the explant into soil? How do you release the agar from the roots without damaging them? I haven't tried it yet, but the looks so fragile.

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I can put roots on a month old cutting in about 10 days if aggressive and transplanted into another vessel, 2-3 weeks au natural. Or a week if removed from the agar, shaven, and dunked into a clean rockwool cube with clone gel(grr, hormones) like traditional clones.  That's the way I work with them when I do. I've no need to root anything in vitro at this time, Chuting was my challenge for a long time (au natural) but I do cultivate little tubes of  root material samples. When I do transplant a rooted plant stuck in agar to the grow room I harden it off for a few days and put the whole rinsed gently agar/rootball into my cubes/plugs and water normally. I do leave a little clear cup over them for a few days, and not so much water in the substrate. Those poke through like normal clones in a few days

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I can put roots on a month old cutting in about 10 days if aggressive and transplanted into another vessel, 2-3 weeks au natural. Or a week if removed from the agar, shaven, and dunked into a clean rockwool cube with clone gel(grr, hormones) like traditional clones.  That's the way I work with them when I do. I've no need to root anything in vitro at this time, Chuting was my challenge for a long time (au natural) but I do cultivate little tubes of  root material samples. When I do transplant a rooted plant stuck in agar to the grow room I harden it off for a few days and put the whole rinsed gently agar/rootball into my cubes/plugs and water normally. I do leave a little clear cup over them for a few days, and not so much water in the substrate. Those poke through like normal clones in a few days

Thanks for the tips...gives me some other options. I am curious what you mean by "shaven." Do you shave the outer layer off of the stem butt to expose a different type of tissue? My explants usually have a sort of bulbous formation on the base of the stem. 

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Just a quick shave of the end of the stem, to remove any dead material and to cleanly expose a fresh slice of cambium layer.

 

when I culture normally its just a pea size explant taken form the donor usually in a new growth area meristem. A piece of leaf is good but I find a slice of the "V" is hardier to withstand my rigorous sterilization with bleach/alcohol/bleach. A good segment of the sample dies and needs to be cut off before a final rinse and subsequent culture.  This multiplies/divides until I snip from it again or transplant into another vessel. I don't root/chute those samples ever, instead I treat them like typical mom donors.

 

when I place developed cuttings into culture, ones with leaves/stems visible, maybe a few inches tall. Most of the time a small callus forms sometimes at the end of the stem that's unneeded to propagate. I shave that off with some material and discard.  I ignore the agar and some is removed before cloned traditionally. 

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Grassmatch, I'm thinking of getting one of those ultrasonic cleaners that you had recommend in the thread. If I normally soak my explants in bleach for, say, 10 minutes, should I use the same concentration and times in the ultrasonic, or shorter? I don't have much contamination issue, 10% maybe, and would like to pare that down a bit.

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after trying a bunch of different cycles and % I settled on a 5 % bleach solution sonic'd for 3 minutes, then a 3 % (91%) alcohol), bleach again, then sterile water for 3 minutes also. 10 minutes might be ok in your cleaner though, using scrap trimmings and keeping notes is good. 

 

by the way....your 10% failure rate is lower than mine in summer months.  I've  seen a seasonal infection rate and find it acceptable. Mid winter I can get to 10% best, 20 worst, but summer is 20 best and 30+ worst.  Recall I don't use a flow hood or any moving actively filtered air. It is scrubbed and ionized big time during all other times just not during culture play.

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mrdizee ,,

when its time to culture again gather up your favorite strain calyx from the flowering/pre flowering and use that sample. I swear its the most difficult, because like many other things, the rewards are strangely different. Not in the finished product, but the experience of getting her there. They are delicate flower bits, be gentle, maybe whisper to her a little. :)

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The leaves coming out of the callus are regenerated from the callus, not from a cutting. The original cutting was just a small section of leaf that grew into the callus from which the new leaves came. They are very slow growing at this point and it's the only piece of callus that has grown leaves. The other calli just sit there, expanding, some green, some yellow. 

 

So, the idea with the battery/LED in the envelope would be for future shipping purposes? 

 

How long does it generally take for enough roots to form to move the explant into soil? How do you release the agar from the roots without damaging them? I haven't tried it yet, but the looks so fragile.

If you figure out how to get roots on that chuting sample I'll walk you right through the hardening off and transplanting stages.

You leafy sample will need a new home soon in any case, I suggest rooting it..... they get freaky difficult later with just leaves and use up resources quick once they reach the stage in my experience. she may have slowed for this reason? don't be afraid of excising another sample or two first before transplanting if you need the genetic store, otherwise I'd root it now while its thriving shiny win!

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Colchicine is a toxic chemical that is often used to induce polyploidy in plants.(crocus leaves, etc) Basically, the colchicine prevents the microtubule formation during cell division, thus the chromosomes do not pull apart like they normally do. The end result is a cell that now has double the number of chromosomes that it would normally have. If this cell divides again in the future, then the doubled number of chromosomes are passed to the offspring cells. Plants that have more than the normal two sets of chromosomes are termed “polyploidy” in general, although specific names are given to the certain chromosome numbers (e.g. tetraploid or 4N plants have four sets of chromosomes).

 

Polyploid plants are generated in an effort to create new plants that have new characteristics. Sometimes the polyploidy plants are sickly and not viable, but sometimes the polyploid plants have larger leaves and flowers. Orchid growers will often sell polyploidy plants that are larger or have larger flowers. Often the polyploid nature of the plant is included in the cultivar name (G. species ‘big flower 4N’). Colchicine is also used to try to make fertile hybrids between species with different numbers of chromosomes.

 

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Colchicine is a toxic chemical that is often used to induce polyploidy in plants.(crocus leaves, etc) Basically, the colchicine prevents the microtubule formation during cell division, thus the chromosomes do not pull apart like they normally do. The end result is a cell that now has double the number of chromosomes that it would normally have. If this cell divides again in the future, then the doubled number of chromosomes are passed to the offspring cells. Plants that have more than the normal two sets of chromosomes are termed “polyploidy” in general, although specific names are given to the certain chromosome numbers (e.g. tetraploid or 4N plants have four sets of chromosomes). Polyploid plants are generated in an effort to create new plants that have new characteristics. Sometimes the polyploidy plants are sickly and not viable, but sometimes the polyploid plants have larger leaves and flowers. Orchid growers will often sell polyploidy plants that are larger or have larger flowers. Often the polyploid nature of the plant is included in the cultivar name (G. species ‘big flower 4N’). Colchicine is also used to try to make fertile hybrids between species with different numbers of chromosomes.

Cut and paste source; https://www.growery.org/forums/showflat.php/Number/196213

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I have piles of useful information like this one in data files. I saw the remainder of the article was not as useful as the part I posted. any feeb with google can type in two words of a paragraph to find the source, glad you were figured out how to do it.

If you have nothing related to Tissue Culture kindly move along, perhaps troll another topic/poster you love, and give me a break.

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I have piles of useful information like this one in data files. I saw the remainder of the article was not as useful as the part I posted. any feeb with google can type in two words of a paragraph to find the source, glad you were figured out how to do it.

If you have nothing related to Tissue Culture kindly move along, perhaps troll another topic/poster you love, and give me a break.

When you quote Professor Cahill word for word you should provide the source. Otherwise it looks like it's your original thinking. He teaches a great course and deserves full credit for his work. 

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I boil my well water in a tea kettle, then sterilize it with tools in the pressure cooker.

No issues yet. I used to sterilize bottles of RO water but tried this a few years ago and was fine.

 

 

How long did your explant take to develop all those chutes from callus ?

I originally took the leaf cutting last October and have divided the callus several times. Once I put it into the container you see in the photo it took a couple weeks for the leaves to show up. Yesterday I put it into rooting formula.

 

Attached is another piece of callus that was actually in a shoot formula, but just keeps getting larger. After a month it has grown to present size from a pea size. Going to take a sample and put it into another shoot formula to see if I can get it to respond in a favorable way. 

 

post-39662-0-62882500-1457477167_thumb.jpeg

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