Node and callus tissue culture pictures

[Palito_Blanco]

I know this tread is over 2 1/2 years old. Your work looks really nice. Two green thumbs (in vitro) up. Would you be able to update us with more recent tc work.



5 years ago I was living in a non cannabis friendly area of the States. I wanted to stay connected with as much horticulture techniques as possible. Because I knew cannabis is where I wanted to spend my life working. And really took to the idea of tc. I did set up an area in my home for tc using miniture roses as my working material. I was succeful in virto but never ex vitro. I would lose them in the hardeing off stages.

I'll be setting up a tc lab here in Uruguay for cannabis. This time around I found a tc course that will guide me thru the entire process even setting up a proper lab. Really excited and cant wait to get started.

Thank you again for sharing your work.

Palito

 

[GrowerGoneWild]

Hi Charles,
do you have any tips regarding sterilizing tip shoots for culture? I have played around with TC a few times using a pressure cooker to autoclave my materials, water, and medium, and working under a makeshift protective hood and frequently sterilizing my tools in 99% iso. For the tip shoots I have been doing a 10% bleach solution rinse for 10min, followed by a ppm rinse (pure plant preservative).

However despite my careful attention to sterility, I have lost most of my cultures to contamination after a few weeks. I was wondering if there are any method or processes that you can recommend regarding sterility, thanks!

I know this is an old post, but might be helpful for other TC people.

I'm using a 2 step sterilization procedure, after having high contamination rates too. And my bad for not using a fancy air hood.

http://www.sigmaaldrich.com/technical-documents/protocols/biology/explant-sterilization.html.

But to cut to the chase.. A quick rinse in 70% ethanol. (note the short exposure time for explants with ethanol) Rinse in sterile water, Bleach soak in 10% then rinse, then I go for a final rinse in H202 then follow up with sterile water. Multiple sterilization solutions instead of just the 10% bleach.

Your final rinse could have PPM in it if desired.

I think my biggest problems with contaminated cultures was yeast or spores getting into the culture. So I spent more time into making a still air "hood" instead of using a container set on its side. It was closer to a glove box.

And since I suspect spores were getting in, my last sterilization step was hydrogen peroxide 3% that is a known antifungal. And since chlorine is damaging to plant tissue, I think H202 could neutralize residual chlorine.

 

[whatAriot]

excuse me, I'm new to TC and would like to clean up some genetics. Does anyone do this successfully? i see most of these posts are old. thank you

I know this is an old post, but might be helpful for other TC people.

I'm using a 2 step sterilization procedure, after having high contamination rates too. And my bad for not using a fancy air hood.

http://www.sigmaaldrich.com/technical-documents/protocols/biology/explant-sterilization.html.

But to cut to the chase.. A quick rinse in 70% ethanol. (note the short exposure time for explants with ethanol) Rinse in sterile water, Bleach soak in 10% then rinse, then I go for a final rinse in H202 then follow up with sterile water. Multiple sterilization solutions instead of just the 10% bleach.

Your final rinse could have PPM in it if desired.

I think my biggest problems with contaminated cultures was yeast or spores getting into the culture. So I spent more time into making a still air "hood" instead of using a container set on its side. It was closer to a glove box.

And since I suspect spores were getting in, my last sterilization step was hydrogen peroxide 3% that is a known antifungal. And since chlorine is damaging to plant tissue, I think H202 could neutralize residual chlorine.

 

[resin_lung]

Some have been successful to a certain degree. What works for one should not be expected to work for another. Some don't seem to respond well and they may never. The basics are out there to be studied and found easy enough. Contamination is gonna be an issue. It always is. Technic is very important but its usually not enough. If success is 1 for every 100 technic and the bare minimums are enough. If you want real success you might have to spend some money. It's getting cheaper but..... it's not cheap. Laminar flow

 

[resin_lung]

Double post