Or_Gro’s Tissue Culture Journal

[Or_Gro]

Beginning day 2:



On contamination watch, 2-3 weeks til ready to root....

 

[Or_Gro]

Beginning day 2:

View attachment 4349752 View attachment 4349750 View attachment 4349749

On contamination watch, 2-3 weeks til ready to root....

Again: this is an inefficient approach for rooting cannabis, rooting can be done with a lot less foreplay using scissors and an aerocloner in ~10 days....

This method is being demonstrated, to show the sterile technique, and to test effectiveness of a less expensive hepa fan rather than an expensive full-blown laminar-flow setup....

Next up, real tissue culture: taking slices of these samples, turning them into miniature plants, rooting them in tube/jar, then storing some plants and transplanting others to normal growing environment.

 

[pthobson]

Nice man.

 

[Or_Gro]

Nice man.

Thanks man!

 

[led1k]

Ok, almost there...

Decided, wtf, i have a sterilizer/pressure cooker, why not use it?

Pressure cooker (PC) sterilizing process:

1. Get all your sh!t together, and prepare.

2. Fill PC with water, to appropriate level, insert wire rack/trivet to keep contents out of water.

View attachment 4348870

3. Add ~10mL (~3/4-1” depth) nutrient solution in each test tube, place cap on tubes but do not tighten (unless you want a f’n mess), put tray with nutrient tubes in PC.

View attachment 4348875

4. Loosely wrap scalpel, forceps, scissors, paper towels in aluminum foil, and place in PC.

View attachment 4348871 View attachment 4348872 View attachment 4348873 View attachment 4348874

5. Put cutting plate in PC.

View attachment 4348877

6. Put lid on PC, tighten appropriately, open steam valve, turn on heat.

7. When steam starts coming out of valve (mine takes about 50 mins to get up to steaming speed), let run for 5 minutes, then close steam valve/put on steam weight, when pressure builds to 15psi (mine takes about 45 minutes to build), run for 20 mins.

8. After running for 20 mins @15psi, shut off heat. Let cool to room temp (overnight), DO NOT OPEN until you have moved pressure cooker into your sanitized “lab”.

9. When ready to do your tissue work, open PC, arrange items for good workflow.

Very nicely done. I was thinking about what happens when the PC cools ... If in non hepa air will it possibly suck in contamination?

 

[Or_Gro]

Very nicely done. I was thinking about what happens when the PC cools ... If in non hepa air will it possibly suck in contamination?

First, always let it cool naturally...never release pressure until opening.

Theoretically, the cooling process can suck in air....but I have found that my pot still has a little pressure at room temp, after overnight cooling...mine is made for this purpose, others may not hold a seal at room temp....

No harm, tho, letting it cool appropriately in your “cleanroom”...

 

[Or_Gro]

Day 3: No evidence of contamination....yet.



Running lights 24:0, @ 40-45 ppfd (1x96elite + 1x65w incandescent), to maintain steady heat @ ~75-77F, rh 50-60%...

 

[Or_Gro]

Another use for plants with balls:

https://www.slideshare.net/mobile/manteshmuttappagol/anther-and-pollen-culture

 

[Or_Gro]

Or how about artificial seeds from tissue culture?

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3550375/pdf/12298_2009_Article_8.pdf

 

[Or_Gro]

Just a great f’ing link:

https://biocyclopedia.com/index/biotechnology_methods/tissue_culture_techniques/sterile_methods_in_plant_tissue_culture.php

 

[led1k]

Just a great f’ing link:

https://biocyclopedia.com/index/biotechnology_methods/tissue_culture_techniques/sterile_methods_in_plant_tissue_culture.php

Excellent! No tote for a glove box? Their cardboard version would work perfectly well.

 

[Or_Gro]

Excellent! No tote for a glove box? Their cardboard version would work perfectly well.

Cardboard works for shrooms...sterile tek is the key..

That link is pure gold... all kogs in there...

 

[Or_Gro]

One of the common themes i’m reading in diff plant tissue culture (PTC) descriptions is use of 18:6 light schedules.

I’m used to running 24:0 during seedling/areocloning, and will be doing so here; so, i’ll find out if it makes a diff..

 

[led1k]

One of the common themes i’m reading in diff plant tissue culture (PTC) descriptions is use of 18:6 light schedules.

I’m used to running 24:0 during seedling/areocloning, and will be doing so here; so, i’ll find out if it makes a diff..

15:5 was mentioned somewhere... Maybe a faster day/night cycle brings them along faster?

 

[Or_Gro]

15:5 was mentioned somewhere... Maybe a faster day/night cycle brings them along faster?

You do one with some lightsout, we compare?

 

[Or_Gro]

Day 4, no contam....startin to get my hopes up on that hepa fan’s ability to do the job in a 4x4....next stop a larger bathroom setup....

 

[Or_Gro]

Testing some small scale humidifiers, here’s first one:

Pulled stem apart and soaked tampon for 10 mins


Doesn't look like much


Put it in this monster tote


Starting temp & rh


5 mins later


30 mins later


Winner, winner, chicken dinner!

Manage rh level with a timer...

Here’s a 2’x3’x4.5’:

 

[Moabfighter]

This very much reminds me of taking mycology slants. Neat.

 

[Or_Gro]

This very much reminds me of taking mycology slants. Neat.

Cells are cells...

 

[Moabfighter]

Cells are cells...

Indeed. I’ve wondered this years ago I swear. Frickin cool to see someone trying to innovate it. Awesome job man. I’m actually in the market for a laminar flow hood, just trying to find one local ish for about 500 bucks......

Sending out a 2ml vial ready to plug in dirt whatever and go..... this IS what’s up man. Neat. Wow.