Tissue Culture/Micro Propagation Grow from Scratch

Looks good canndo, I really hope those beans pop for you. I can't find anything in my notes on the ratio, but I'm pretty sure it was around 1.5-2% strength. I know it was a little less than regular drug store peroxide. I would think you'd want to run a little on the higher side, to ensure sterilization. I doubt 3% would do any harm, it can't hurt to try.

Me to Wol, I mean I'm not asking too much am I? I got some peroxide and will try it next batch. I wonder how it would do on plantlets - probably too brutal eh?

well sir damn subbed up for the rest of what ever you do. as i have been loooking into this, only skimmed through this over the past hour and learned a lot tomorrow I will actually read it. But damn thank you for this.

canndo said:

Me to Wol, I mean I'm not asking too much am I? I got some peroxide and will try it next batch. I wonder how it would do on plantlets - probably too brutal eh?

Click to expand...

Eh, I don't think it would harm them if you kept the concentration relatively low. I've used it as a foliar spray in the past when I had fungal issues.

These are the best of the lemon skunk, the others are in good shape but not as nice as these. It is obvious now that they are gaining robustness, I suppose is the term. No sign of roots but it is still early.


Here is my Afghan Kush uber-mom. She is on this day 7 months old, I have gotten many cuttings from her and she survived the thick gel, the excess TDZ and all the other abuse I have thrown at all of my tests. This is the one I wanted to try flowering with. The crown of the "plant" is so thick with shoots you can't see any of them. I want to see how she does at a year.

Now look at these, check out the thickness of the leaves, they are both indicas but then my Afghan Kush is supposed to be as well, isn't it? and the leaves are very narrow. I know that we can't apply the same understanding of apparent differences between normal sativa and indica and.. these plantlets but they don't all look the same when in the micro state. The middle picture was a cluster over two inches across - it about filled the baby food jar. I was sure that I had arrived when I pulled this one out for transplant - I figured I'd have at least 10 shoots. No,well yes, there were 9 shoots but this one was hit by hyperhydrosis.

Vitreous plants. Something happens to them and they get glassy, change color slightly and change in consistency to a fragile crumbly plastic like mess. Even just brushing them with a scalpel will break the leaves or stems off. If you are lucky you can break all of the vitriotic portions off and still be left with some viable plant material as I did here. If not, the plantlet will survive but not grow. I have read reports that you can revert some plants some times to their original state with paclobutrazol but I have not been able to have this work in the tests I have done. (admittedly a limited grid).

I also tried to use Paclobutrazol in my media as a preventative with no sucess. I believe, as I have been saying, that the problem is putting a plant which really doesn't like high humidity in a jar with 100 percent RH. I don't intend to grid this out but I have learned (as we all do when working with plants), what seems better than what. When I see the first evidence of hyperhydrosis I transplant the plantlet and most times this sets the process back. With this plantlet however, I just couldn't tell if it was the strain itself or hyperhydrosis. I don't have specific stats but I get hit with this stuff maybe as much as 10 percent of the time.

Now given that I have a 20 to 30 percent loss rate at the establishment phase and a 10 percent loss rate at the multiplication phase due to contamination, if I could save that other 10 percent I'd be a lot better off. (addressing contamination loss rates - I fully expect my rate to go down now that the room is dedicated to this enterprise). I am hoping that vented caps will help. I am also expecting that I should be able to vary the RH in the jars to what ever I wish - if they are vented with large enough holes.
La
These test explants are from Skywalker. I have to continue to vary my sources of cuttings in order to see if there is any variation.
Here is my soon to be modified super lab - 50 here, 12 up top and another 10 below. 70 moms or 70 clones depending on how you look at it. Mind you I am counting only one plantlet in each jar and that isn't the case with many of these. After the establishment phase one could easily have 3 plantlets in each jar. Now that is 150 clones on one shelf.

While I have put a great deal of work and time in this endeavor if I were to consider only what it took to get to this point i.e. what it would take to go from zero clones to this point, I would think that it takes me about an hour to make the media, another hour to sterilize it and about 10 seconds per jar for establishment and 30 seconds per jar for multiplication. More or less. I'd have to count the sterilization of water, the sterilization of cuttings and such so add another hour or so.

That Afghan-Kush is crazy. I can't believe the difference in appearance from that and the lemon skunks to the thick leafed pic below it. It looks like you have things rolling along nicely. I really want to see some roots, and how difficult it will be to harden the plantlets off so they can be grown in a standard setup. Eagerly waiting.

And finally for the day. WHAT the HELL is up with my seeds? I can't tell because of course I can't SEE them anymore, but the ones I can see have done nothing. My control shows not a single seed opening. I know it is early for the control and I have not put them in the best situation with regard to heat. I just don't want to set the mat/controler/containers up. Did I get a whole bag of bad seeds? Gee I hope not.

canndo said:

And finally for the day. WHAT the HELL is up with my seeds? I can't tell because of course I can't SEE them anymore, but the ones I can see have done nothing. My control shows not a single seed opening. I know it is early for the control and I have not put them in the best situation with regard to heat. I just don't want to set the mat/controler/containers up. Did I get a whole bag of bad seeds? Gee I hope not.

Click to expand...

Oh yeah! The seeds! That's nuts that none of them have even sprouted. What are your temps like?

My temps are low, mid 60's in the day and high 50's at night. I don't want to run my room heater for just this and as I said, I havn't put up my heating pads yet. I was simply going to be patient. If I don't see anything in a few more days I'll set them up. I wanted to ease up on my utilities as my other enterprise hit me pretty hard - one of the reasons I don't do a traditional grow is the cost of electric.

canndo said:

My temps are low, mid 60's in the day and high 50's at night. I don't want to run my room heater for just this and as I said, I havn't put up my heating pads yet. I was simply going to be patient. If I don't see anything in a few more days I'll set them up. I wanted to ease up on my utilities as my other enterprise hit me pretty hard - one of the reasons I don't do a traditional grow is the cost of electric.

Click to expand...

I'd say that's the issue with the seeds, from my experience. Ideally the temp should be a constant 75 degrees for optimal sprouting conditions.

Ok, so even my control is not working, it has to be as Wolverine says, heat. I got a couple more heat mats - the ones with the sensor control, I figure I can use them for hardening off - if we ever get to that point. Still, what is it with me and seeds? Nothing, not in the control and not in the sterile containers.

Reports - my contamination rate has fallen as I predicted since there is not steady stream of purple spores or green spores being spewed into the room. the prefilter in my HEPA system had a forest green sheen to it. So my last starts seem to have a loss rate of 5 percent. Not so good with the vented jars - I lost two out of 12 so far.

These are only a few days old. Dank is using non-vented boxes and I like the idea of more realestate but not for starting single plantlets. I lose one I lose them all - except that I have had some sucess recently with rescuing a single one if I catch the contamination in time and if I happen to have a fresh, unused jar of media hanging around. I am hoping along with an orbital tilt plate (more on this soon), I can get a new fridge. If I do I intend to see if I can store media in the fridge to see if the chemicals can be made to last longer. I tried freezing the media in the same interest - don't bother, it doesn't work.

Rooting? nope. What is happening is that the shoots are elongating and consolidating, they don't have that precarious, "if you use me I will die" look about them. I think it has to do with the GA3 ratio. It may be reasonable to alternate formulas or introduce a "consolidation" formula somewhere in the plant's life. I have been thinking about moms a lot here. I know that I can quickly induce shooting but do I always want the mom shooting if I am interested in having the plant in a genetic library? I could simply place the jar in the fridge but plants like to grow, even tiny things such as these. I am thinking that a cell elongation and multiplication formula that does not induce shooting might be a good thing to have around.

If I don't see roots in another week or so I am going to add activated charcoal to see if I can yank the cytokinins out of the plant. I hate waiting for this stuff, I have more things I want to know but I can't continue any longer without seeing (and proving) roots form.

I am also back on the artifical seed route again. I now believe it is possible for a lay person to create artificial seeds and those seeds should be able to "germinate" in non-sterile conditions. Hell, it might be that the only way I can get germination is to make my own seeds. Doing this should eliminate the randomness of phenotypes.

Nothing. No roots, and no sprouts. I'm starting the sprouting again.

Gotta be the seeds. I've never had a problem germinating seeds unless they were very old or improperly stored.

I am now on my second batch of controls, they are in a wet paper towel sandwiched between two plates and set on top of my fridge. I will next try to soak them for 24 hours and keep them on one of my mats. This is getting frustrating. So far as the roots are concerned? nothing yet. I'll give them another week and then proceed to another formula.

holy shit, i just landed in geek heaven for marijuana growers. totally scribed and gunna have to read from start, cuz i read the last two posts and im dumbstruck. Keep this thread going!!!

Just an update. No roots and no seedlings. Other chemicals have been ordered but I mostly need caps. I have to expand again and do not have the containers (the caps) I need to do so but I can't see paying a buck a cap, there has to be another way. The larger containers, though valuable are over 4 dollars each, considering that if I continue this, I will have need for hundreds if not thousands of jars, I have to come up with another solution.

I lost 8 jars to contamination from the original 70 and every one of the rooting experiments show solid little plants...with no roots, I am not worried. I soaked some seeds in water for 24 hours and placed them between napkins and plates and put them on my fridge. Still nothing. I have to get the control working before I will extend the experiment to the sterile tubes as it is a bother to fill them with STG and sterilize them for seeds that are evidently dead. I have 9 Hog seeds but I don't know if I want to sacrifice them for this.

What you see above is three new cuttings from a few days ago, they are three different strains of unknown origin. I want to see if there will be problems establishing various strains as I discussed earlier. They look pretty good.




There is a duplicate here but these are all our original lemon skunk cuttings, scroll back if you wish and look at how they have progressed, remember that these are all cuttings off of cuttings that have grown themselves. I could easily take three cuttings off of each of thse but of course, we are waiting for them to root instead. Had I been interested in continued multiplication of the Lemon Skunk I think I would have, 60 of them at this point, and shortly thereafter, 180. I can no longer claim to be doing all of this under 17 watts and in under 4 square feet. I added two more shelves. Now I can potentialy have 210 moms under 60 watts of light in 12 square feet (unless I place more than one plantlet in each jar - othewise we have the potential of having 630 "moms" or "clones". Notice what I've been talking about, they seem to be solidifying, growing more robust.

I think we can now safely say that an amateur, with a modicum of experience in some other form of aseptic conditions for the cost of a couple of electronic balasts, can establish and multiply almost any strain of cannabis.

Another pass at rooting should work, I am unconcerned.
I believe I have an automated solution for hardening off. (but I am concerned)

I have yet to:
Establish using a seedling
sex an unrooted plantlet
sex a rooted plantlet
Root
Harden
create an artificial seed


Tending to parameters of light and heat are on the agenda, I have said that I suspected that the plantlets were not getting enough light so I propped a few of them up a few inches on a box. They grew greener than the controls and they seemed to grow more quickly but this is subjective and counter to everything I know about these proceedures. It is still cool here and I am still hesitant to bump the temperature of the room up with a 1000 watt room heater. My lab runs a rather large HEPA filter continuously with the exhaust vented and deflected in order to minimise rapidly moving air. I blocked the central heating vent to the room so if the rest of the house is being heated, dust would not be thrown into the room. Because of that, the room's temperature is very different than the rest of the house, the only way I can manage it is with an electric heater. We have one of the highest electrical rates in the country here and I HATE paying the higher tiered bills. But my experiments show that optimum temperature is between 80 and 85 degrees.

Heating mats do not work as I had hoped because I cannot manage them. I have a thermostat connected to the mats but I don't know where to put the sensor, so far, placing it on top of a vessel simply keeps the mat on all the time.

That is some hard work my friend ...!!
Wow props.
I love your pictorial.
+Rep

Puffer Fish said:

That is some hard worm my friend ...!!
Wow props.
I love your pictorial.
+Rep

Click to expand...

I agree ...this is one awesome dude and thread huh puff

Namaste'

canndo said:

Just an update. No roots and no seedlings. Other chemicals have been ordered but I mostly need caps. I have to expand again and do not have the containers (the caps) I need to do so but I can't see paying a buck a cap, there has to be another way. The larger containers, though valuable are over 4 dollars each, considering that if I continue this, I will have need for hundreds if not thousands of jars, I have to come up with another solution.

I lost 8 jars to contamination from the original 70 and every one of the rooting experiments show solid little plants...with no roots, I am not worried. I soaked some seeds in water for 24 hours and placed them between napkins and plates and put them on my fridge. Still nothing. I have to get the control working before I will extend the experiment to the sterile tubes as it is a bother to fill them with STG and sterilize them for seeds that are evidently dead. I have 9 Hog seeds but I don't know if I want to sacrifice them for this.
View attachment 1563463 View attachment 1563464 View attachment 1563467
What you see above is three new cuttings from a few days ago, they are three different strains of unknown origin. I want to see if there will be problems establishing various strains as I discussed earlier. They look pretty good.


View attachment 1563465View attachment 1563466View attachment 1563468View attachment 1563470View attachment 1563469View attachment 1563462

There is a duplicate here but these are all our original lemon skunk cuttings, scroll back if you wish and look at how they have progressed, remember that these are all cuttings off of cuttings that have grown themselves. I could easily take three cuttings off of each of thse but of course, we are waiting for them to root instead. Had I been interested in continued multiplication of the Lemon Skunk I think I would have, 60 of them at this point, and shortly thereafter, 180. I can no longer claim to be doing all of this under 17 watts and in under 4 square feet. I added two more shelves. Now I can potentialy have 210 moms under 60 watts of light in 12 square feet (unless I place more than one plantlet in each jar - othewise we have the potential of having 630 "moms" or "clones". Notice what I've been talking about, they seem to be solidifying, growing more robust.

I think we can now safely say that an amateur, with a modicum of experience in some other form of aseptic conditions for the cost of a couple of electronic balasts, can establish and multiply almost any strain of cannabis.

Another pass at rooting should work, I am unconcerned.
I believe I have an automated solution for hardening off. (but I am concerned)

I have yet to:
Establish using a seedling
sex an unrooted plantlet
sex a rooted plantlet
Root
Harden
create an artificial seed


Tending to parameters of light and heat are on the agenda, I have said that I suspected that the plantlets were not getting enough light so I propped a few of them up a few inches on a box. They grew greener than the controls and they seemed to grow more quickly but this is subjective and counter to everything I know about these proceedures. It is still cool here and I am still hesitant to bump the temperature of the room up with a 1000 watt room heater. My lab runs a rather large HEPA filter continuously with the exhaust vented and deflected in order to minimise rapidly moving air. I blocked the central heating vent to the room so if the rest of the house is being heated, dust would not be thrown into the room. Because of that, the room's temperature is very different than the rest of the house, the only way I can manage it is with an electric heater. We have one of the highest electrical rates in the country here and I HATE paying the higher tiered bills. But my experiments show that optimum temperature is between 80 and 85 degrees.

Heating mats do not work as I had hoped because I cannot manage them. I have a thermostat connected to the mats but I don't know where to put the sensor, so far, placing it on top of a vessel simply keeps the mat on all the time.

Click to expand...

Did you start with a different seed source or are you using seeds from the same batch?

Wolverine97 said:

Did you start with a different seed source or are you using seeds from the same batch?

Click to expand...

Same batch Wolverine, a friend handed me a big old bag - I think there is a picture of it somewhere. If this last test goes south (or doesn't go anywhere) then I will have to conclude that he must have done something horrible to those seeds. I think I am showing that I am a patient guy in this thread but this seed thing is trying my nerves something horrible. I want to try this method out very badly, propagating from seedlings is a sure way to go (aside from being unable to confirm phenotypes).

I also figure that it would help others who want to start doing this by avoiding the difficulties of serility. I have demonstrated that you can sucessfuly do propagation in a kitchen without a hood of any kind if your technique is down and you use a decent biocide and are willing to take some initial contamination hits.

We know that even if our sucess rate is as little as 25 percent in the establishment phase multiplication will make up for the loss in a matter of three weeks. But, I believe that going directly from seedling will give the newbies confidence. I don't want anyone to refuse to do this sort of work because "it's too hard" or "you have to be too clean" or "it is too expensive" and a part of that is GETTING THOSE DAMN SEEDS TO SPROUT.