Tissue Culture/Micro Propagation Grow from Scratch

I say growth tips....

Namaste'

The session went fairly well. I didn't over do the bleach and let them soak in water. I also let some of the buds grow in a bag for a bit so that there will be fewer contaminants on them. I couldn't sleep so I figured I would fill everyone in on the progress. Now the temperature his up in the high 80's. While this is good for the little plantlets it is making me miserable. I figure we are down to a week before rooting.

I think I can play expert on cutting ex plants now. Perhaps soon I will write a little primer on the subject, it is different that simply taking clones in the normal fashion.

And I will make another list of the auxiliary things you might want to have. I don't know how many are still watching, but what would you like me to explain or demonstrate?

Night !

Ok,


My best friend is my mag stirrer. I can use it to make media easier than anything. You just set the thing up, add the liquid, add the sugar and the MS and adjust the Ph to 5.80. Don't use paper strips and be exact, I have found that a tenth to either direction inhibits growth. The thing about the stirrer is that it is easy to adjust the Ph because a drop is dispersed in seconds.

My other best friend is my ultrasonic cleaner. A drop of detergent in with 5 percent bleach in the cleaner is about as thorough as you can get. The vibrations get inside the tiny hairs and it strips off of those persistent little bubbles that tend to stick to the cutting even when it is shaken or stirred.

Get a Big pressure cooker, they aren't that expensive and you can put enough jars of water to sterilize, plus your instruments, plus your napkins and work plate. If you are doing media, the bigger one will do 40 at a time easily (getting them out of the cooker efficiently something I haven't yet mastered).

A good bottle brush is very nice.

A good garbage disposal is tremendously helpful. You put a lot of this gel down the sink, unless it is spun up fine it will clog your pipes and you really don't want that. Many bag the stuff up and throw it out rather than risk such clogs. I would rather keep the disposal running as I dump jar after jar into it's spinning maw.

nitrile gloves

A wonderful little device is an ultrasonic denture cleaner. It is a battery operated sub-ultrasonic cleaner. It sets up standing waves in the liquid that is great for initial washes of dirty plantlets. I have also used it for bleach and alcohol washes to great effect. If I were to want to do this on a shoestring I would keep this over the full ultrasonic - AND the mag stirrer. They are something like 10 dollars each. So I would consider getting one for each phase of sterilization (well that really isn't the best word for it).

crawling insect killer - spray it on your counters once every two or three weeks

Some lab glass - a liter and a two liter and a half a liter Erlenmeyer. A couple of graduated cylinders.

Premoistened antiseptic wipes - handy for spills, quick wipedowns and such. Get a container for a buck. Speaking of which, make the 99 cent store your second home for a bit if you want to do this cheap. I laugh and laugh when I see admonitions from the experts -


"oh no, you need thousands and thousands of dollars for a lab and ultra sterile conditions, you can't do this, it is too expensive" I've had people tell me that I would need 10 thousand dollars just to start.

Of course the prime big boy toy is a decent laminar air flow hood, except when you open a contaminated jar in front of it. I have shown though and will continue to show that this is not necessary. All that you need is a good biocide and good technique and a willingness to accept a surprisingly small increase in losses. This, if you decide to get it is about $500 for a little one - and $1500 for a single person nice one that doesn't cramp your style.

A series of RACKS - racks that hold your jars or tubes or tubs. Very handy but not necessary

If you are doing this from scratch you will need another set of items - graduated pipettes, a good milligram scale, a measuring boat, a fine paintbrush (when you are working in thousandths of a gram, you can easily leave a significant portion of your powder in the boat unless you brush it out.

I didn't put this in the essentials I don't think, I'll check but you MUST have distilled water. Again, when you are working with something that works in the 1 to 10 ppm range you don't want RO water that has residuals of - 10 to 15 ppm. You want something with nothing in it but... water. I am going to finish my transfers in a few minutes. I believe, since I am in the mood to write, that I will describe the exact method of cutting and sterilizing plantlets - with pictures. Won't that be fun?

Illumination said:

what he said^^

Click to expand...

Wait a minute you guys - I asked you what you wanted me to explain and you both said "yes please". Now I am confused.

First you have a branch, if you look closely you will see that the growing tips are a bit yellow, this is because I put the tops in a bag for a few days. I don't think this will affect the growth too much. The second picture is some of the plantlets. The smaller ones are tips the larger ones are lower auxiliary. I would have wanted for there to have been a little less growth of the aux buds as they shot out enough where I will not be able to use the larger stem as base. The next two pictures are all of the explants merrily spinning in 5 percent solution.


The first plant in this series is an orientation shot. Note that this is the plant from the last series of implants - if you have been tracking this one you can see the enormous spurt of growth, this is the one that has convinced me to take a different attitude toward cuttings, size, source and the like.

The others are all the best lemon skunk spawns. Again, each of these entire jars started out as a single, slim tip cutting about the size of a pencil lead and about 3/4 inch long. We have easily tripled the biomass of the original bit of material. Were we to opt to continue to propagate, I would go another two, perhaps three weeks this time and take at least 9 new cuttings from each jar. We are now at 45 clones in under two months. One more pass given another 3 months would be 135. Total cost to me is about 30 cents per jar every 3 weeks and 20 watts of light 16 hours a day. .32 kwh in my neighborhood costs a little more than 3 cents.

canndo said:

Wait a minute you guys - I asked you what you wanted me to explain and you both said "yes please". Now I am confused.

Click to expand...

Here, wait, maybe this will help. YES. PLEASE.

D

canndo said:

Ok,


My best friend is my mag stirrer. I can use it to make media easier than anything. You just set the thing up, add the liquid, add the sugar and the MS and adjust the Ph to 5.80. Don't use paper strips and be exact, I have found that a tenth to either direction inhibits growth. The thing about the stirrer is that it is easy to adjust the Ph because a drop is dispersed in seconds.

My other best friend is my ultrasonic cleaner. A drop of detergent in with 5 percent bleach in the cleaner is about as thorough as you can get. The vibrations get inside the tiny hairs and it strips off of those persistent little bubbles that tend to stick to the cutting even when it is shaken or stirred.

Get a Big pressure cooker, they aren't that expensive and you can put enough jars of water to sterilize, plus your instruments, plus your napkins and work plate. If you are doing media, the bigger one will do 40 at a time easily (getting them out of the cooker efficiently something I haven't yet mastered).

A good bottle brush is very nice.

A good garbage disposal is tremendously helpful. You put a lot of this gel down the sink, unless it is spun up fine it will clog your pipes and you really don't want that. Many bag the stuff up and throw it out rather than risk such clogs. I would rather keep the disposal running as I dump jar after jar into it's spinning maw.

nitrile gloves

A wonderful little device is an ultrasonic denture cleaner. It is a battery operated sub-ultrasonic cleaner. It sets up standing waves in the liquid that is great for initial washes of dirty plantlets. I have also used it for bleach and alcohol washes to great effect. If I were to want to do this on a shoestring I would keep this over the full ultrasonic - AND the mag stirrer. They are something like 10 dollars each. So I would consider getting one for each phase of sterilization (well that really isn't the best word for it).

crawling insect killer - spray it on your counters once every two or three weeks

Some lab glass - a liter and a two liter and a half a liter Erlenmeyer. A couple of graduated cylinders.

Premoistened antiseptic wipes - handy for spills, quick wipedowns and such. Get a container for a buck. Speaking of which, make the 99 cent store your second home for a bit if you want to do this cheap. I laugh and laugh when I see admonitions from the experts -


"oh no, you need thousands and thousands of dollars for a lab and ultra sterile conditions, you can't do this, it is too expensive" I've had people tell me that I would need 10 thousand dollars just to start.

Of course the prime big boy toy is a decent laminar air flow hood, except when you open a contaminated jar in front of it. I have shown though and will continue to show that this is not necessary. All that you need is a good biocide and good technique and a willingness to accept a surprisingly small increase in losses. This, if you decide to get it is about $500 for a little one - and $1500 for a single person nice one that doesn't cramp your style.

A series of RACKS - racks that hold your jars or tubes or tubs. Very handy but not necessary

If you are doing this from scratch you will need another set of items - graduated pipettes, a good milligram scale, a measuring boat, a fine paintbrush (when you are working in thousandths of a gram, you can easily leave a significant portion of your powder in the boat unless you brush it out.

I didn't put this in the essentials I don't think, I'll check but you MUST have distilled water. Again, when you are working with something that works in the 1 to 10 ppm range you don't want RO water that has residuals of - 10 to 15 ppm. You want something with nothing in it but... water. I am going to finish my transfers in a few minutes. I believe, since I am in the mood to write, that I will describe the exact method of cutting and sterilizing plantlets - with pictures. Won't that be fun?

Click to expand...

For getting them out of the pressure cooker maybe you could find a similar sized pasta cooker (straining pot fits inside stock pot) and use the strainer (the bottoms are flat) to hold your jars, then you just lift the whole thing out when done.

One the water: my RO water comes out around 5ppm initially, then works its way up to 9 or so when the membrane is close to the end of its life. Do you think that would work?

1. Wrap your instruments - a couple of scalpels, a few sets of tweezers, a pair of scissors in some aluminum foil.

2. Fill at least 5 decent sized jars with tap water (although I use RO because my tap water is so absolutely crappy), cover then (foil if you must)

3. Cut some slices of paper napkin, or paper towel into squares, lay them flat and wrap that in aluminum foil as well.

4. Put all these things in your pressure cooker, bring to temp and pressure (15 lbs) and cook for 20 minutes. Allow time for everything to cool.

5. Make yourself some 5 percent solution of unscented bleach with RO or better water - it doesn't need to be sterile, the bleach will do that just fine. You do this by using your graduated cylinder and pouring 10 ml into the cylinder. Then you top it off to 200, finally add two drops of unscented detergent. The detergent breaks the surface tension of the water so many of those micro bubbles will go away and it allows closer contact with the bleach water. Depending on the method you use, you might want 400 mls.

6. Make yourself some 10 percent solution as well. This time you use 20 mil bleach, top it off to 200 and add your detergent. Depending on the method you use, you might want 400 mls.



7. Select the branches you wish to collect explants from.

8. Inspect them for the cleanest and most healthy nodes

9. Rough cut those nodes, trim all fan leaves even the little ones - be very careful not to damage the growing tips.

10. Rinse them in cool tap water. Really rinse them. If you suspect that the are inordinately dirty you can place them in a jar with a screen on it and run a steady stream of tap water into the jar for any number of hours.


Or you can place them in something like this ultrasonic cleaner with clean tap water and a drop or two of unscented detergent. Let that run for half an hour or up to a few hours. What we are attempting to do is dislodge any dirt particles. The water does not yet need to be sterile. Save your sterile water for the later processes.





Now in the same jar you will be doing all of these procedures place all of your explants and pour several ozs of 70 percent into the jar - swirl it for five or six seconds. I MEAN it. Never more than 10 and you even need to allow time for your pouring the stuff out and rinsing. What we are trying to do is dissolve some of the sticky stuff that is all over these plants - the isopropal will kill the plant in short order and we are going to be doing this again.

Pour the isopropal out and immediately pour in cool water. I now prefer sterile water Now swirl that, and dump it out. Repeat the rinse again.

200 mils of your 5 percent bleach should be plenty if you are using ultrasonics. If you are using a stirrer, do it again. If you are shaking it, either way will do.
Shake, stir or standing wave ultrasonic for 15 minutes. Ultrasonic for 4.

While you are doing that prepare another solution of 10 percent bleach. (that's 20 mills topped off to 200 - don't forget the detergent. When the plantlets have reached the required time Dump out the old water, replace it with the 10 percent. Shake, stir or standing wave for 10 minutes. Ultrasonic for 2.

Dump that water out

Now rinse with sterile water.
Dump
Rinse it again
Dump
Rinse it again
Dump
YES, Rinse again.
Dump.

Now pour 70 percent isopropal in your system and shake, stir or standing wave for 25 seconds - DO NOT use isopropal in your ultrasonic device. So far as I have experienced you will have wasted your effort and killed every plantlet if you do but DO use the isopropal - this is a final aseptic wash and it does a pretty good job.
Dump that out.
Rinse with sterile water
Dump

Yes, actually do it 8 times. Leave your plantlets in the water and make sure that the container is covered. We don't want you to have gone through all that trouble only to invite spores to float onto the top of the water.

Now you have sterile explants in a covered container of sterile water.

If you find that most of your explants turn brown or black consider dialing back your second isopropal wash. If you find that most of your explants turn white or yellow consider dialing back your second 10 percent wash.

If you get contamination on the sides or in the "leaves" of the plant consider bumping up your first bleach wash a couple of minutes. If you get contamination at the base, within the gel, consider pushing your second bleach wash up a minute or two. I don't often mess around with the isopropal washes but be my guest. I also don't much change the bleach percentages - too many variables but in the past I've gone as high as 15 percent - it doesn't seem to do anything more dramatic than bleach the plant. The point is very simple. Kill all the nasties on the plant without killing it.

Now look, if you have read this far, good for you, I know you can do this. It looks like a whole whole lot of work and steps but it isn't really. Let's break it down.

sterilize your instruments and a lot of water for rinsing
make some fresh weak sterilizing solution and fresh strong sterilizing solution.
wash all of the free dirt off of the plants
use a solvent to rinse that characteristic marijuana sticky stuff off of the plant (this is essential to your sucess)

use the weak bleach solution to work it's way into the crevasses of the plant
Use the strong solution to wack the more hearty more easy to reach nasties
Now nuke the plant briefly with super strong alcohol for anything that may remain
And finally get ALL of the soap, alcohol, and bleach out of the plant.



Ready to do this thing now? This is actual, I want to see if this can be done without a hood or glovebox, just in the open air in my kitchen.


It is important that you arrange your setup. Fluidity and efficiency is key to this procedure. Notice that the explants are in the jar on the left, my instruments are on the right in a jar full of denatured alcohol or, in this case 91 percent isopropal. I have two tweezers and two scalpels, I alternate. After each procedure I place the instrument back in the jar and take the other. This allows more time in contact with the antiseptic. I do not use flame. Any excess alcohol on the blade or tweezers is blotted off briefly on the sterile towel which is on my ceramic, sterile workplate (99 cent store sushi plate - great).

I have found that the majority of infection is in the fibers at the end of the cut places. If we use a sterile scalpel and slice off just a bit of the already bleach damaged end we improve our results.



So, what we do is slice a bit off of every portion of the plant. If we can, we cut just a bit off of the still attached leaves being ever so carful not to cut too closely.

You are working to gain the most surface area exposed to the gel and the least otherwise so cut accordingly.

You should do as many plants as you intend to place in ONE jar at first. I recommend that you don't do more than one plant for each jar until you get the method down. Work quickly, exchange your instruments often but not so often that it interrupts your work. Find a rhythm. I will fish out three likely candidates from the water jar, place them on a new place on the paper towel, exchange tweezers, make all the required cuts. Then I will replace the scalpel, exchange tweezers and begin inserting the plantlets into the jars.

Align the plantlet as close to parallel to the tweezers as you can.
Make the plantlet an extension of the tweezers. It isn't all that easy but you can not properly place the plantlet if it is at a right angle to the tweezers. I have always found this to be difficult, you must try not to have to adjust the angle of the plantlet after you have already picked it up. If you must touch the plantlet to something in order to adjust it's position, use the inside rim of the jar itself.

I am finding that depth of media is important. A little extra depth - perhaps as much as 3/4 inch may use more media than we would like but it allows us to use longer stems, have more surface area exposed to the media and compensate for weak gel. Stiff gel doesn't seem to release nutrients as well as soft but soft will allow the plant to sink or fall over. If you make your "stem" just a bit longer and allow enough gel to support that longer "stem" you are in better shape.



If you want to look like a pro, you must pick the gel jar up with your left hand, open the cap while holding it, with the fingers of the left hand and hold it at an angle so the opening is not facing directly up. There is a reason for this as well, you shouldn't switch hands, you shouldn't leave the cap open longer than necessary, you shouldn't ever set the lid down.


Now insert your plantlet with your right hand on the tweezers.



Close the lid the same way and set it down.



Views from the top of the jar - do your best to never have the jar open and upright but if you don't position the cutting correctly, then take the time to do so, it is more likely that the cutting will not grow correctly if it is not positioned than your getting contamination. Anything submerged in the gell will not grow. Try to get the growing tip or crux just slightly above the gel, it will drown if it is not high enough. If it is too high it doesn't seem to grow as quickly. Multiple growing points, if close enough together will give you a jump on getting new shoots for later cuttings.

You can place the cutting as a "log" parallel to the surface if the growing tip points outward enough. I like to but the bottom edge of the cutting down on the base of the jar if possible, or just slightly above that point.









Now ensure the lids are tight, label or mark them if you have not already done so and place them in their spot under a light, in temperatures of the low 80's. Keep your eye on them for the first week. You will find smears of cream colored bacteria, or tiny threads of contamination or discoloration in some of them, when you find them take them away and wash them down the sink. What you have left will likely live. We will go through taking cuttings from explants next time.

Wolverine97 said:

D

For getting them out of the pressure cooker maybe you could find a similar sized pasta cooker (straining pot fits inside stock pot) and use the strainer (the bottoms are flat) to hold your jars, then you just lift the whole thing out when done.

One the water: my RO water comes out around 5ppm initially, then works its way up to 9 or so when the membrane is close to the end of its life. Do you think that would work?

Click to expand...

No sir. I am sorry but even at 5 you are competing with the hormones. I don't know what is in your water and it may well be just fine but why take the chance? Pay the buck and a quarter a gallon.

canndo said:

No sir. I am sorry but even at 5 you are competing with the hormones. I don't know what is in your water and it may well be just fine but why take the chance? Pay the buck and a quarter a gallon.

Click to expand...

Yeah, not a problem. It would have been nice though if the $800 Culligan system I have was capable.

canndo said:

1. Wrap your instruments - a couple of scalpels, a few sets of tweezers, a pair of scissors in some aluminum foil.

2. Fill at least 5 decent sized jars with tap water (although I use RO because my tap water is so absolutely crappy), cover then (foil if you must)

3. Cut some slices of paper napkin, or paper towel into squares, lay them flat and wrap that in aluminum foil as well.

4. Put all these things in your pressure cooker, bring to temp and pressure (15 lbs) and cook for 20 minutes. Allow time for everything to cool.

....

Click to expand...

You must spread some Reputation around before giving it to canndo again.

Epically awesome post man. This page will be bookmarked for sure.

View attachment 1528768The seed experiment is now a failure - View attachment 1528767 That is as far as they get. The ones that didn't germinate during the first week never did, the ones that did germinate never advanced beyond this stage. I tried the bubble idea wolverine. Swirling does not set bubbles, shaking at just the right moment does but most of them tend to rise to the top and I think they are too big. Any suggestions on anything here are welcome and if it is feasable and reasonable in any sense of the word I will do my best to try.

So. This is my third attempt. The first was with full sugar, NAA MS and gellzan - PH 6.5. Don't hate me for using hormones, I had lots of spare media and I just put it into containers and slipped some seeds in. The second was full sugar (30 gr per liter standard - my establishment formula uses more now), full MS and gellzan - PH 6.5.

This one was the Chinese protocol. Half sugar, half MS PH 6.8. They used full light - theirs worked mine don't.

This is not a controled experiment, in fact little of this now is, I left those behind with the basic protocols for callus, establishment and multiplication because without a decent floor of dependable growth I couldn't move on to actually proving the viability of this form of propagation. I had to actually know which and in which ratios were the most effective combinations. In so doing I got a feel for hormone balances within our plant of choice. Now before anyone starts marveling at my magnificent ingenuity and years of dedicated work, be aware that all you really have to do is draw a grid , mix a bunch of chemicals according to each section of the grid, cut up a bunch of plants and watch. It doesn't take that long to begin to figure out what works and what doesnt.

I can't draw up a grid for the variables here, drop seeds into each permutation of variable and then sit back and watch. I don't have enough seeds and frankly, although desirable from an esthetic point of view, it is not imparative that I germinate in gell media.

I figured I'd take this public because I had never seen it done and believe me I looked. I also knew that there were some smart people here who would be willing to give me ideas. I took this public before I had a proper rooting protocol for Cannibis Sativa because I felt comfortable with the hormones and people are rooting Cannibis every day. There should be no difference between the rooting of a macro plant and the rooting of a plantlet.

So, back to the seeds.

Sugar
minerals
light
gell agent
oxygen
temperature
treatment of the seed
PH


So we can learn quite a bit simply by adjusting the PH of some rock wool and cranking that up. I am thinking that we need a baseline. Today I will get some rockwool, put it in some vessels and put a few seeds in there. No sterilization of media or seeds.

I would like to know what else people wish to see explained about what we are doing here. Anyone?

A primer on Growth hormones then?

canndo said:

A primer on Growth hormones then?

Click to expand...

my friend that would be awesome...as well as the explanation of each one's exact causal effect

Great thread, I just found it...I could be of some help if you need any. I have done this many times...but never with weed itself. Of course it is easier in a sterile lab environment. One thing I noticed si that your cuttings seem to be to big; you only need thin slices of the apical meristems which are where the nodes and alternate branches form. You could take severall cuttings from the ones you have use.

Also, you need horomone additives like IAA, some form of auxin/cytokine. Also, if you use cubes instead of test tube the plants can actually grow out big enough to be transplanted. This is mainly suited for leav tissue culturing. you can literally use one finger of a leaf and wash with alcohol and bleach solutions as u've done and disect it into tiny square pieces as you would in skin tissue culturing. Photos are better then words...

DankBudzzz said:

Great thread, I just found it...I could be of some help if you need any. I have done this many times...but never with weed itself. Of course it is easier in a sterile lab environment. One thing I noticed si that your cuttings seem to be to big; you only need thin slices of the apical meristems which are where the nodes and alternate branches form. You could take severall cuttings from the ones you have use.

Also, you need horomone additives like IAA, some form of auxin/cytokine. Also, if you use cubes instead of test tube the plants can actually grow out big enough to be transplanted. This is mainly suited for leav tissue culturing. you can literally use one finger of a leaf and wash with alcohol and bleach solutions as u've done and disect it into tiny square pieces as you would in skin tissue culturing. Photos are better then words...

Click to expand...

Thanks for your input and I welcome all input. Firstly though, I have been trending toward larger explants because they have a quicker start. I started with what you describe and waited weeks for anything to happen.

If you check the early posts you will see that I do indeed use Cyt/Aux combinations along with other growth stimulators - I have worked through a dozen different combinations in order to get cannibis to grow well enough. I have yet to get a leaf or a callus to grow a shoot and studies I have read show sucess in the 1.2 percent range. I have no probem abandoning one technique for another so long as my I continue to get closer to my goal so no leaves for me when I can as easily just use original stem cuttings. So far as the boxes are concerned I would love to use them. I use test tubes only for seeds, baby food jars and tubs seem to work pretty well. Because I am going for multiplication so far, I would prefer pretty much the size I have. If and when I get to actually using lots of plantlets for standard growth I will begin to try larger vessels. Thank you for your interest in my progression and especially thank you for any tips you might have.

What is your experience with activated charcoal?

I have been interested in using activated charcoal and have been reading up on it but haven't actually used it in any microproagation experiments as of yet...

Dank? Are those boxes vented and if so how? I am certain that I need to modify the humidity in my jars, or whatever I use. Where did you get them?

woah man one day ill experiment with something like that

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...