In vitro micropropigation

[rootsnshoots]

Great thread! Are you forming a callous first and then going the way of roots and shoots (no pun intended!)?

Nope going for shoots here first. Actually I don't really know what I'm doing at all. But hey sure is fun.

 

[Ythor]

Nope going for shoots here first. Actually I don't really know what I'm doing at all. But hey sure is fun.

It's tough to see from the irregular texture of the glass, but from the first two images, it looks like you already have callus, along with 1-2 shoots. You were starting with leaf material, and not nodes, right? If this is the case, you probably have callus with shoots.

Your contamination issues will almost always come from insufficiently disinfested starting material. Indoor grown plants will be cleaner than those from outside, due to environmental contamination. Wash in running water, cut your explants, and disinfest: ethanol, peroxide, chlorine (DCCA is better than sodium hypochlorite). The bleach is the important step, but some people add one or both (alcohol and peroxide) to improve the chances of getting rid of any spores.

 

[rootsnshoots]

Ythor thanks for the tips! The plants are grown indoors. I forgot about the tap water rinse first, that'll help a lot. The one growing looks like kind of a gnarly knotty stem with probably 4 shoots coming off of it. Is that stem callus? I'm pretty sure that one was a nodal segment from the top of a plant. Basically I topped a plant and then peeled that top bulbish new growth apart and used the bottom of it. The big leaf that grew fast and big but contaminated, was the tip of one of those leaves.

Can you get growth from just cellulose?

Whats the best to use for explants?

I'm almost positive the contamination came from lack of cleaning the explants. But I did have one that contaminated the agar away from the explant. Looks like Trichoderma. That one was probably a floaty..... Shmuv...

 

[Thoth]

Good stuff bro! great to see how your experiments are coming along. Really makes me want to give it a crack, though would need some serious investment into equipment before I could do that.

Keep us updated!

 

[rootsnshoots]

Good stuff bro! great to see how your experiments are coming along. Really makes me want to give it a crack, though would need some serious investment into equipment before I could do that.

Keep us updated!

DO IT! Surely grandma's gotta presto you can borrow? ;)

I got some water cooked today so I'm going to make it a priority to get down at work tomorrow. I'll take tons of pics and do a step by step write up.

Ythor, ANY suggestions, pics, ideas, criticism, please feel free. I know if you wanted to show everyone yer skillz you'd have your own thread... But this one is as much yours as mine. And anyone with questions or suggestions, links please post! I would like to keep this thread open, friendly, and a good guide for anyone wanting to try, and as a good document for checking my mistakes.

 

[Ythor]

Can you get growth from just cellulose?

Whats the best to use for explants?

I'm almost positive the contamination came from lack of cleaning the explants. But I did have one that contaminated the agar away from the explant. Looks like Trichoderma. That one was probably a floaty..... Shmuv...

Dead, dry cellulose will not be propagatable, no. For most plants, you want meristematic tissue- stuff that's growing the fastest, usually a growing tip of some sort- and that includes floral material. For most plants, this includes nodes; not cannabis, best as I know. The literature seems pretty clear in that most people use leaf material.

Contamination away from the explants is usually airborne (or coming from the hands, that sort of thing). Disinfestation of tissue just shy of killing it may be required; there are all sorts of tricks and techniques, most of which involve fairly complex liquid techniques. However, one fairly simple trick is to use an ultrasonic generator- the cheaper, the better. More powerful units will also destroy plant tissue. So, while the plant matter is in a disinfection solution, that container is placed in the ultrasonic cleaner (usually a cheap jewelry cleaner) for a few seconds- enough to help collapse air bubbles and enhance penetration.

 

[entropy99]

Great thread Rootnshoots. Micropropagation is absolutely fascinating.

Have you found your experiments to be costly so far, including the cost of equipment, hormones, etc.?

Have you tried using growing tips as material?

 

[rootsnshoots]

Dead, dry cellulose will not be propagatable, no. For most plants, you want meristematic tissue- stuff that's growing the fastest, usually a growing tip of some sort- and that includes floral material. For most plants, this includes nodes; not cannabis, best as I know. The literature seems pretty clear in that most people use leaf material.

Contamination away from the explants is usually airborne (or coming from the hands, that sort of thing). Disinfestation of tissue just shy of killing it may be required; there are all sorts of tricks and techniques, most of which involve fairly complex liquid techniques. However, one fairly simple trick is to use an ultrasonic generator- the cheaper, the better. More powerful units will also destroy plant tissue. So, while the plant matter is in a disinfection solution, that container is placed in the ultrasonic cleaner (usually a cheap jewelry cleaner) for a few seconds- enough to help collapse air bubbles and enhance penetration.

I figger no dead stuff. Was just wondering if taking the inside of the stalk will be more contam free, as its not exposed to air. I know when cloning from mushrooms you take your culture from the inside of the stem. As it is contaminant free.

The tdz article used nodal segments with great results

I have a three head ultrasonic puck laying around actually. Thing will kill the shit outta some shit. I put a Tupperware lid over it one time. I see the lid start melting, turn it over and it was unaltered. It burned the top side first. Kinda cool. So maybe just wave your plant material over the transducers a couple times. I see what ya mean by the air bubbles. I added a drop of dawn to the bleach solution. Supposedly that helps? Or pftt calls for tween.

Liquid techniques.. please enlighten us!

Slurry in a hurry? Blend up a bunch of clean leaf/plant material in a sterile water solution. Then inoculate the tiny bits?

 

[rootsnshoots]

Great thread Rootnshoots. Micropropagation is absolutely fascinating.

Have you found your experiments to be costly so far, including the cost of equipment, hormones, etc.?

Have you tried using growing tips as material?

Thank you, and I agree!

No not really. I already had a lot of this stuff but if you started from nuttin.

45$ Hepa "type" filter from wally world as of last time I checked. They sometimes have a true hepa and you can replace my models with one... But mine does the job. I will say this style "glove box" has gotten a lot of doubt and negative responses by people.. usually people that have not tried it. The thing really should not work in theory. It's hard to maneuver in. I've simplified part of the problem by using the jars and sterilizing the media in the jar vs pouring sterile media.

100-300$ pressure cooker. Scored mine on craigs for 100$ 21.5 quart all American.. don't skimp on yer cooker. Shell last you the rest of your life.

100$ hormones ms agar It makes a ton. You use none. Stores well? I'm hoping

25$ torch. I use a plumbers torch outside of the shmuv to sterilize tools, then cool them in sterile water or on the agar before handling the explant

50$ cleaning supplies, gloves, 55gal clear drum liners, tweezers,

10$ 12 jars.

5$ micropore tape. Wally world, walgreens

FREE$ TYVEK. The post office has tyvek envelopes. Punch a hole in the lid of your jar, cover it with 3 layers of tyvek and tape it down with micropore tape :) this allows for gas exchange in the culture vessel and filters out any bad guys.

Ythor when thawing my stock tdz. Can I just thaw a little bit and use that or should I thaw the whole jar, stir and then measure?

 

[monkeymun]

Keep it Rootnshoots, loving this thread!

 

[rootsnshoots]

Here's the sterile run. ill post the media prep and tool/water prep next time i do it.

Use the smallest room you have that closes but gives ample room the work, like a bathroom. you can put shelves or wood down on the bathtub to make a table. Wipe down the walls and floor with a disinfectant. OUST that mofo and close the door. Come back in and set up the shmuv 10 mins later.

Blow up the shmuv. Use the filter to inflate the bag. Don't flap it in the room like you would if you were using it in a thrash can. I tape it down to the table, then tape it to the filter. Inflate it and then cut the arm holes. tape the arm holes to prevent further tearing from moving your arms. I cant find 55 gal clear bags so this is smaller. and it sucks.. sourcing:mad:

Alcohol the shit out of the inside of the bag and filter, as well as anything and everything that goes into the box. Jars, tools, your gloved hand and arms, Every time I pull my arms out of the box, I mist them with alcohol before they go back into the box.

Here we are set up. 8 vessels awaiting inoculation. I like to loosen the lids slightly before getting them saturated with alcohol. It is hard to crack em in the box without ruffling the hell out of the bag. I get em popped loose and then douche the shit out of em with alcohol and slide them through the arm holes.Also I have tweezers sterilized and wrapped in aluminum foil. and an alcohol soaked paper towel.



Lets go hunt a pretty young mama.. Take some cuttings..



Then I brought them to the sink. Cut off pieces I knew I didn't want and ran them under the tap for a bit. Scrubbing with my clean fingers.

Then I cut off pieces of leaf and nodal segments with a clean razor blade over the 10% bleach sterile water solution. I squirted a little 3%h2o2 in there too. Didn't measure the amount probably should have.

I am not sure if i should be putting the cuts into the wash jar in open air? Or if i should put all the explants together in the jar, or should i wash them individually?



Soaked the cuts for 10 mins shaking/ swishing the jar every couple minutes.



Blast that baby with iso and slide er in the box.



Transfer the cuts from the wash jar to the sterile water jar with sterile tweezers, then to another sterile water wash. swishy swishy but steady not to disrupt the bag. All arm movement must be minimal and smooth to reduce back draft into the bag.



Remove the band from the explant jar but leave the lid on. Grab a culture vessel and remove the band and leave the lid. I have another culture vessel "on deck" next to the jar im inoculating, which i will set the band on. Then slide the lid half way off the explant jar and pluck out a cut with sterile tweezers. Lift (as minimal as possible) the lid off your culture vessel and drop in the cutting, cap and ban. Repeat..



inoculated 2 jars of .1,.2,.3,.4 mg/l tdz one nodal one leaf each.
no Gibber this time.



here is a jar i had set on the side throughout the entire sterile sesh. As kind of a test to see if and how much contamination id get. I've done this before and had plates not contaminate most often. but then i had extra cuttings so i thought hey toss em in..



Now we wait....:D I'm stoked on the clearer-er-ish jars. its hard to get clear pics of it but looks better in person.TRUST ME!:p





update on the one that popped on the first run.

 

[Ythor]

I figger no dead stuff. Was just wondering if taking the inside of the stalk will be more contam free, as its not exposed to air. I know when cloning from mushrooms you take your culture from the inside of the stem. As it is contaminant free.

The tdz article used nodal segments with great results

Maybe I'm thinking of a different article, but was that the Chinese one where they used TDZ on nodal segments? Never had much luck with nodal segments myself.

For clean stuff- seeds that are still enveloped in plant material should be sterile- not that the seeds themselves are tough to disinfest- but that's not really cloning in that you have no idea what you're starting with.

If you're willing and able to cut down tissues to get to the meristem, sure- you can go that route; still need to surface-disinfect, and cut down into them to get what you want. Some people do carnations (Dianthus) that way, and it takes a skilled operator to get down to the meristem and do it cleanly.

For the melting part- if you don't want to melt and re-freeze your large stock solutions every time, I recommend aliquotting your stock solution into the microcentrifuge tubes, a little more than 1.000 ml per tube. Normally I use 1.010 mL (about as high as a pipettor will go), so that I don't have to recover 100% after I thaw them. You can't just partially melt a 50 ml centrifuge tube of liquid and hope it's homogeneous; normally solutes (in this case, the TDZ) are the last to be incorporated into the ice, unless it's flash frozen at great speed.

 

[rootsnshoots]

I don't mind melting and re freezing. As long as its not bad for the solutions or hormone. Ok I just let a little melt and sucked it out with a syringe. I'll let it fully melt next time. And probably transfer it to more smaller jars.

 

[squiggly]

Because of the nature of impurities when they are a part of a crystal lattice (solid or liquid, depending on composition of the mixture) it is important for ice to be completely melted before a sample of the proper concentration can be retrieved.

Some of your hormone is almost guaranteed to be locked up in the ice still if it hasn't all frozen.

Generally, cold won't hurt chemicals (it tends to stabilize them, actually)--so I wouldn't be too worried there, although there are exceptions to this.

 

[rootsnshoots]

Soo the jars I did probably don't have any or much less tdz than I thought. I did the same thing last time maybe that's why the one with a "strong" amount worked best. Sucks cause that means my stock solution is probably more than .1mg/ml now

 

[bioguy]

I have a couple of thoughts that might help somehow

1) I have sprayed H202 directly on my plants at 15% concentration (I use a 50/50 mix of 30% and tap water to clean my res and could not resist spraying a plant once). This is beyond the 10% usually used for sterilizing and could be done ahead of time to prep the explant

2) It seems like the cleanest tissue might be a fresh root tip. A clone machine with sterile water in a hepa box might be a way of producing clean material. Even the root as it breaks free of the rockwool should be really clean

3) If its a seed strain it could be sprouted in the hepa box very easily.

Rootsnshoots, are you in Colorado? You said MMJ but that could be a lot of places.

 

[rootsnshoots]

I've thought a lot about using roots. Makes me wanna fire up the old aero cloner. I'd imagine just runnin h2o2 through it for a bit and then cut bits of roots off.

If and when I have another flowhood things will go much smoother. You just need to get one cut to shoot and then you can go aseptic to aseptic with ease.

Yep the 303

 

[digdugdank]

Awesome thread, loads of great info! Roots, you have done your homework well, and I will be picking your brain for knowledge, as well as squiggly, dankherbs, and the rest of the farm.:) I will be reading with interest, and hopefully be able to add a few of my follies to the table! Keep up the good work! Thanks, digdug!

 

[bioguy]

I read that initial work with micro propagation on cannabis led to hermies and the pheno's were not accurate...they eventually figured it out. It makes me think that the "power washing" of punked out genetics might not be pure "broscience". If past results led to plants unlike the explant it may be that micro propagation maintains the genotype not the phenotype...this could be responsible for the power washing. I am speaking in theory solely based on the fact that plants have been different from the explant.

Remember that the DNA content is only part of the game...DNA regulation/ is a major part of what we see when we look at a plant. i.e. the punked out clone may still have all the DNA needed for the original quality but its not being enforced/regulated. The more I think about it IBA and the other hormones we use may have even turned them off (big time speculating here). It makes sense if you consider/assume that the micro propagation hormones cause the hermies and lost phenos. What I am trying to say is that if a plant can get new traits (hermies and lost/new phenotypes) from micro propagation hormones then it does not seem crazy that those same hormones would do the same thing (or something similar or equally weird) to our clones.

I guess I am just not ready to give up the hope the same way the first researchers did not give up when they got hermies and lost phenos

Perhaps I'm just an optimist

 

[pork]

got a full micropropigation kit in my hands a couple weeks ago...going to give this a try and see what this broscientist (physicist turned hobby chemist and botanist) can find out for myself...certainly worth a shot.