Tissue Culture/Micro Propagation Grow from Scratch

very cool, i've heard about this before but never actually seen pics of someone doing it.

DankBudzzz said:

Ummm no, they are not ventilated.

The horomones and properties of the agar allow the plant to be grown in a relatively anaerobic environment...They are completely sealed and never opened until analysis as they don`t contain any antibacterial in it. They are very susceptible to mold and fungus...

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I have found that the plant suffers from a variety of vitirious conditions when it is constantly in 100 percent humidity. Studies indicate that I can aleviate some of this with a slightly lower RH. I do use chemicals that keep the gelling agent fairly clear of contamination. I have also found however that this protection tends to disapate after a few weeks. None of those chemicals however, will inhibit contamination on the plant itself. There are some pictures I have floating around this site of what the plant looks like when conatminated with one of the "fungi perfecti"

skiweeds said:

very cool, i've heard about this before but never actually seen pics of someone doing it.

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I have looked and never found anyone chronicling their efforts in micropropagation of cannabis. Stick with us as we explore what happens next. Thanks.

I have another experiment running, the gradual increase of TDZ. Now I know the limit of effective dosage of this chemical. Note the browning of leaves and the leaf curl as a result of the overuse of this.

The last two shots are of healthy moms, I am ready to take cuttings again but will not do so in favor of rooting. The count is still the same on the lemon skunk. The experiment was to take one set of cuttings only and then to root, the plantlets look fine but a little brown - there is no leaf curl. I expect they will do fine in the next phase. I am due to recieve the chemicals I need today. I will set them to root in the morning. I am again short of vessels and I can't seem to get any more magenta caps so I am going to larger vessels. I have settled on IBA and NAA as my rooting compound. Let us see how that one works. I will start another round of cuttings just in case so that the experiement won't have to wait.

Common Auxins:
Dicamba An herbicide that I find particularly useful in the generation of callus in C.S.
2,4-D A weed killer. I’ve not used it
IAA It is likely that you all have used IAA as it is one of the more common hormones in rooting dip and powders – it works moderately well with C.S.
IBA is another very common rooting hormone used frequently. It is more stable than IAA being less sensitive to light and heat. This is the one I have selected to use, the Chinese study uses it and I have every confidence in it as it is used frequently in woody plants.
NAA is also used to produce roots and callus. I find NAA to be very useful as a balancing hormone in C.S.\

Comon Cytokinins:
BAP, one fo the most used growth hormone. It works with C.S.
PBA Used for auxiliary shoot production. It works with C.S.
2iP Another commonly used hormone that works with C.S.
Kinitin is a growth regulator that is isolated from DNA. IT works somewhat with C.S.
Zeatin is or was extracted from corn endosperm. Although it is a common Cyt, I found it worked poorly with C.S. in the proportions and amounts I tried.
Thidiazuron is a defoliant that has cyt properties I have found this to be a very good Cyt for C.S.

I can only speak to general actions of each of these classes of chemicals because they act differently on different plants and they act very differently in conjucunction with each other and with other growth inducing chemicals,

In general, tissue cultured with auxins CAN cause cells to grow large but not divide. When cultured with Cytonkinin alone there is little or no effect.

So we talk about apical dominance. This is a theory and I am sure many others could explain it far better than I. Auxins come from the apical bud and travel down shoots to inibit more bud growth so it promotes shoot but inhibits branching. Imagine a christamas tree where the top produces auxins to inhibit the plant from forming more shoot parallel to the main one. As the auxin is diminished futher down the plant, more auxiliary shoots form, so the plant gets wider the further it s from the top. Imagine the same effect on every lateral bud as well.
The point here I suppose is that one can not actually point to a single chemical and say "this chemical does this alone and this or that in combination with another chemical" It is a trial and error sort of thing and I am constantly juggling the ratios for a simple reason. I spoke earlier about a grid that one can use to settle on a particular ratio but in order to triangulate the amounts I have to go in steps. .01 .03 .05 and the like of each chemical. I don't think plants are aware of increments such as these and it could be that optimum performance could be gotten from something like .015 or .033. This would force me to experiment forever before I started actually seeing something green. In this case I have been bumping up the tdz a fraction each time for a portion of my plantlets. I will do the same with the balancing compound. This may not give me an optimum but it will get me closer.

I am very much looking forward to the rooting portion and and rather than make a final cutting,which is what I intend to do further on, I want to give them the best possible chance. I have another question though, and that is that there seems to be some distinction in sativa dominant and indica dominant types. They don't seem to grow the same. Sure, they both work quite well with the solutions I am using but I can't help but wonder if there are differences in their individual needs.

So now we wait for the brown truck.

Yes, I wish I had pictures of them but I had several plants that were contaminated and grew different kinds of bacteria and mold but the plants them selves still thrived. Without antibiotics and antifungal agents in the agarose gel you will definately have problems unless you are in a 100% sterile environment using aseptic techniques. These cubes allow for slight gas exchange, i'll have to check exactly what polymer they are made from but I know it is minimally semi permeable allowing for slight gas exchange but still creating a greenhouse like environment.

Also, it is important to note that in several experiments I've conducted, it is very difficult to find the proper auxin/gibberellin ratio to allow the plants to show considerable growth advantages over plants that were grown in the same media lacking horomones. The general components of the agar and sucrose media provide ample nutrients for the plant to sustain growth. Although many plants showed more of one type of tissue; callus, roots, stems, it is difficult to find a pattern due to different concentrations. It is a very analytical science.

DankBudzzz said:

Yes, I wish I had pictures of them but I had several plants that were contaminated and grew different kinds of bacteria and mold but the plants them selves still thrived. Without antibiotics and antifungal agents in the agarose gel you will definately have problems unless you are in a 100% sterile environment using aseptic techniques. These cubes allow for slight gas exchange, i'll have to check exactly what polymer they are made from but I know it is minimally semi permeable allowing for slight gas exchange but still creating a greenhouse like environment.

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I use a biocide wich is effective on both mold and bacteria.

No brown truck today. Damn. I want to try to save some of the leaf curled plants. I lost my mag stirer element and I had a few of those shipped as well, I don't want to make three different batches without the stirer- and the IBA is on its way with the stirers.

So we wait some more.

I am an idiot. I got up this morning and the package was there, I presume it was there all night. Luckily it was very cool last night or I would have had to do this again. IBA is sensitive to heat and light. I wanted to use it because that is where the Chinese got their best rooting. I think I will use IAA afterward just so I don't have to pay 50 bucks for shipping. Of course if you buy the powder the problem doesn't arise but then I have to make so much stock solution that it doesn't make sense either. The stir bars came as well - should have gotten more as I will surely lose these in a week or so.

Anyway, on we go with the next phase and starting again in case this one doesn't work.

I suppose a page on mixing media is in order.

1. Make a list of all the ingredients you are going to use and check them off one at a time"

NAA
IBA
Biocide
MS
Sucrose
Gel agent
Water

Now be sure you have a pipette for each of the liquid chemicals
use your first pipette and measure out a proportionate amount, that is, if you intend to use 1 mg per liter and your stock solution is 1 mg per ml then you will pipette 1 ml from the stock and into your mixing container.

Do this with all liquids into your measuring column and then carefully top it off with distilled water. The other way of course is to measure out a liter and then REMOVE the comensurate amount of water. I.E. If your total liquid additions are 4 ml then fill your final container with 1 liter and pipette 4 ml out before you put the chemicals into the mix - that way you have exactly one liter.
now add the sucrose and ensure it is all disolved - it takes longer than you will imagine. DO NOT heat the solution.
Check off each step as you go.

I prefer to have everything measured out beforehand.

After you have your full complement of all liquids topped off to 1 liter go ahead and add your sucrose, then see to it that the sucrose is completely disolved. Now add your MS and then adjust your ph to 5.8.

Finally, add your gel agent. No matter what it is, it reacts differently to being heated so you must mix it cold. I have found that after it is mixed it does not stay in mixed form very long, the agent tends to sink to the bottom making your mixture uneven from one jar to the next so I will warm the water while mixing. Note that this still does not dissolve the stuff, only boiling it will do that and I can't see heating it any longer than necessary.

Now you measure out a specific amount to place in each of your vessels. Usually I will use no more than about 3/4 of an inch although recently I have been trying deeper layers for larger cuttings and as I said, I can keep the gel percentage lower if I have more in the jar to hold up the plantlet. This is where your good turkey baster comes in handy. The cheaper ones will dribble out and get slime all over your work surface - you do not want to get any of this stuff on the outside of your jars, it invites nasty things from bugs to mold.

Because I have several different proceedures going on at the same time I may also add just a drop of food coloring so I can tell one mixture from another.

Today. While sterilizing water yesterday I decided to risk ruining a vessel and put some Sure To Grow in it. To my surprise the integrity of the sure to grow was not compromised in any way after 25 minutes exposure to 250 degrees F. Now I don't have to use Rock Wool and I don't have to adjust the PH of that stuff. I assembled a bunch of test tubes and now I start another experiment.

The question now is, how do we sterilize the seeds.

What does ultrasonic waves do to the seeds?
what is imersion in 70% Isopropal do?
How about 10 or even 15 percent bleach?
If I do nothing but simply wash the seeds in detergent, what happens to the contaminants? I am placing the seeds in sterile media that will not support any sort of growth. There is no nutrient in there save the seed and the seed coat itself. If this is true then what are my chances of getting a sterile seedling from the cotlyodons up?

In order to find out I am placing 18 seeds in detergent water ( 5 ml in 100 ml water) and placing them in the ultrasonic system for 3 minutes. I'll rinse them several times and then put them in the sterilized test tubes and STG. I will use this as the baseline as I don't think this sort of treatment will compromise the seed. I am concerned about the size of the test tubes. I filled them to about 1/3rd with STG. Will the stem grow too large? will the root fill the bottom too much in what? 2 weeks?

I'll get pictures of all this asap.

canndo said:

Today. While sterilizing water yesterday I decided to risk ruining a vessel and put some Sure To Grow in it. To my surprise the integrity of the sure to grow was not compromised in any way after 25 minutes exposure to 250 degrees F. Now I don't have to use Rock Wool and I don't have to adjust the PH of that stuff. I assembled a bunch of test tubes and now I start another experiment.

The question now is, how do we sterilize the seeds.

What does ultrasonic waves do to the seeds?
what is imersion in 70% Isopropal do?
How about 10 or even 15 percent bleach?
If I do nothing but simply wash the seeds in detergent, what happens to the contaminants? I am placing the seeds in sterile media that will not support any sort of growth. There is no nutrient in there save the seed and the seed coat itself. If this is true then what are my chances of getting a sterile seedling from the cotlyodons up?

In order to find out I am placing 18 seeds in detergent water ( 5 ml in 100 ml water) and placing them in the ultrasonic system for 3 minutes. I'll rinse them several times and then put them in the sterilized test tubes and STG. I will use this as the baseline as I don't think this sort of treatment will compromise the seed. I am concerned about the size of the test tubes. I filled them to about 1/3rd with STG. Will the stem grow too large? will the root fill the bottom too much in what? 2 weeks?

I'll get pictures of all this asap.

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Oooh, that's a good idea. I hate rockwool with a burning passion, but I hadn't thought of STG. Wouldn't a peroxide solution work to sterilize the seeds? It seems you could run a fairly high concentration without affecting seed integrity at all, then follow with a mild detergent wash to clean away any residuals and rinse as usual. I don't think you can totally sterilize a seed though, as it's formed in a layering process during growth. It seems you would have to grow a plant in semi sterile conditions heavily filtering incoming air to prevent contamination in the first place, and then sterilize the seeds to grow in vitro.

I do think the test tubes could be a limiting factor for you, but I'm eagerly awaiting these results. Good luck canndo.

Peroxide !! good Idea , I will try it next pass. I didn't know that about seeds but that would explain why it is so hard to sterilize them. I don't know if the spores and bacteria will be mobile in this environment. If they will stay with the seed shell or travel up the stem. I am fairly sure they won't colonize. If they stay localized then I can depend upon my biocide - for anything that comes in contact with the media. The problem will come if there is contamination in the bud or upper stem. I know I can wash the plantlet in biocide but the stuff is very expensive. When I get the chance I may experiment with different concentrations of this and see if I can dilute it enough to offer me some savings. It's about a buck a ml.

I managed to aquire some 6 oz vented jars in order to get to the place DankBudzzz was at with his box/vessels. I think they are too big but at least I can try out my theory about venting. NOW, the problem becomes space. I am going to have to mount more lights and retain more shelf space. I now have 70 vessels, all growing one strain or another. It is abundantly clear that shooting and replicating from new shoots works very well. (still, 70 moms in roughly 4 square feet?) I got growth everywhere. The rooting lemon skunks have stopped growing and they seem to be recovering from my abuse (too much GA, too hard a gel and I suspect not enough light). If I am readjusting my shelf space then I may as well start working with light. I will alter the distance between the lights and the jars to three different distances.

The seeds have been in the STG for 3 days now and still no sprouting on the few I can see. I put a sprouting control down today - sprouting in the normal "seed between a wet paper towel" method. I must get something to work and I have to find out what is going wrong.

I forgot to mention in my mixing instructions - what every you do, don't put your gel agent into the mix until you have adjusted the PH. That gel will screw up your ph meter somethig awful.

canndo said:

Peroxide !! good Idea , I will try it next pass. I didn't know that about seeds but that would explain why it is so hard to sterilize them. I don't know if the spores and bacteria will be mobile in this environment. If they will stay with the seed shell or travel up the stem. I am fairly sure they won't colonize. If they stay localized then I can depend upon my biocide - for anything that comes in contact with the media. The problem will come if there is contamination in the bud or upper stem. I know I can wash the plantlet in biocide but the stuff is very expensive. When I get the chance I may experiment with different concentrations of this and see if I can dilute it enough to offer me some savings. It's about a buck a ml.

I managed to aquire some 6 oz vented jars in order to get to the place DankBudzzz was at with his box/vessels. I think they are too big but at least I can try out my theory about venting. NOW, the problem becomes space. I am going to have to mount more lights and retain more shelf space. I now have 70 vessels, all growing one strain or another. It is abundantly clear that shooting and replicating from new shoots works very well. (still, 70 moms in roughly 4 square feet?) I got growth everywhere. The rooting lemon skunks have stopped growing and they seem to be recovering from my abuse (too much GA, too hard a gel and I suspect not enough light). If I am readjusting my shelf space then I may as well start working with light. I will alter the distance between the lights and the jars to three different distances.

The seeds have been in the STG for 3 days now and still no sprouting on the few I can see. I put a sprouting control down today - sprouting in the normal "seed between a wet paper towel" method. I must get something to work and I have to find out what is going wrong.

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I soak all of my seeds for normal planting in a peroxide solution just to be safe. Keep at it man, I'm pulling for you.

Wolverine97 said:

I soak all of my seeds for normal planting in a peroxide solution just to be safe. Keep at it man, I'm pulling for you.

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How long and how strong sir?

canndo said:

How long and how strong sir?

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Actually, I have to look that up. I haven't cracked beans in a while, I've been cloning for so long... I leave them soaking overnight, but I have to verify the strength. I'm thinking it's like 1.5%
I'll be back later after I find my notes, possibly tomorrow depending on whether I locate successfully.

Just a few pictures - the seeds, the seedling test tubes that so far have shown no sprouts. The larger vessesl, some cuttings that yielded 20 clones (try that the conventional way). My ultrasonic cleaner with some seeds in it, and if you will notice, I have already lost a mag stirer bar.

I thought I had a picture of the entire shelf with 60 moms on it. You can see the larger jars with the vented lids - those hold moms as well now.