Node and callus tissue culture pictures

[Charles-scott]

Indeed I got side tracked and did not have a chance to enlarge those pictures my plant techs were calling me to the garden !
Very impressive I can not wait to here more about this process protoplast isolation ? ! looks like we shop at the same stores ha ha
Charles

 

[Sativa Dragon]

Thanks very much times are changing , I am a little bit surprised everyday by the progress that we have made in certain ways I am grateful to be able to do what I am doing I have been put into a position to lead a industry , I have learned to be humble and work as a team player , its been interesting introducing academics to Cannabis and sharing what I have learned over the past 25 years and humbly learning everything I can from those who have lessons to teach me I no longer have to align myself with criminals and people who's primary reason for working with Cannabis is financial I like to think I have grown with the industry from the trenches to the research facility LOL
We have a outstanding research and breeding facility its amazing to lead a team of such special people to create medicine for a entire country .
Charles

I am too considering leaving a great career to follow my heart, it is encouraging to hear your story, I would be grateful for any advice.

Peace

 

[Waldgärtner]

This is very exciting! It's great to see how Cannabis cultivaton is getting more and more advanced.

About the freezing problem:
I'd look into the cryopreservation of apple cultivars.
I know people at universities who are building libraries with hundreds of specimens of heirloom apple cultivars. They use a process to make the plant cells resistant to the freezing process, so they don't get destroyed.
Although they mostly work with the dormant buds I think it should be possible to do the same with Cannabis.

 

[Tonygreen]

Thanks for sharing.

 

[purple_man]

high charles,

glad to see tc being applied in cannabis
last year was the first time i found a paper discussing shoot induction in cannabis callus... (most likely the previous experiments were done with either wrong auxin:cytokinine ratio or a "non-efficient/non-compatible" auxin was used...).

hence no reports of transgenic canna been published yet (the transformation protocols for agrobacterium mediated transformation are there but the shoot induction protocols were missing. obviously leaf discs didn't work to well)

protoplast separation is important if you want to set up a bioreactor and try to automatize the encapsulation of somatic embryos, or plan on cryopreserving the protoplasts (also you guys tried out protoplast fusion of 2 different lines???)

@sam: the reason why the callus died is you have to mix buffers with the callus before freezing -> the buffers prevent cell rapture and modify the speed of "cooling" down of the callus

blessss

 

[Charles-scott]

I wish we had found that paper ! might have saved some time that being said we have figured it out on our own I have to be careful about what I confirm online I have been cautioned not to let my eagerness to share our success become problematic we are still learning the process and establishing proven methods and as i have said before I know enough to be dangerous ! I will update with some pictures as we turn corners and will ask some questions in area's I can share online .
Charles

high charles,

glad to see tc being applied in cannabis
last year was the first time i found a paper discussing shoot induction in cannabis callus... (most likely the previous experiments were done with either wrong auxin:cytokinine ratio or a "non-efficient/non-compatible" auxin was used...).

hence no reports of transgenic canna been published yet (the transformation protocols for agrobacterium mediated transformation are there but the shoot induction protocols were missing. obviously leaf discs didn't work to well)

protoplast separation is important if you want to set up a bioreactor and try to automatize the encapsulation of somatic embryos, or plan on cryopreserving the protoplasts (also you guys tried out protoplast fusion of 2 different lines???)

@sam: the reason why the callus died is you have to mix buffers with the callus before freezing -> the buffers prevent cell rapture and modify the speed of "cooling" down of the callus

blessss

 

[stihgnobevoli]

great thread.

 

[Tonygreen]

What do you use to make callous form a shoot? Do you think it is possible to make clones via root cloning? It works on some plants, I was thinking it might be a good way to go if there was a protocol that would work. Thoughts? Ever been tried?

 

[purple_man]

high bro,

sure you can establish a root"culture" but it is much harder in comparisson to "above ground" tissue, since the sterilization of roots is much harder -> more probs...

after that you can, repique/transfer the root culture to a shoot induction media, or try n generate callus from the roots, and later transfer callus clumps/pieces to a shoot induction media, once you have a shoot, transfer the shoot to a rooting media, etc...

for callus induction check the interwebz, there is a whitepaper for that. establishing a root culture might be harder, cause i never checked for a canna protocol. i did some fragaraia x annansa duch (strawberry) "runners" and root segment culture, the sucess ratio was very low (due to low efficiency of the sterilizing protocol, or the other lab/team members were just not as proficient with sterile working techniques, as needed tehehehe)

blessss

 

[ganjourno]

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!

 

[Charles-scott]

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 am unable to reveal data about compounds or processes , understandably the corporation that employs me would be less than happy if I was to map processes , I have put it on the line a bit to be able to share what I am so please understand that I am simply not able to offer any specifics , there does exist high concentrate compounds that are costly but will help you with your issues !
Sincerely
Charles

 

[purple_man]

hi ganjourno,

if you are looking for a sterilization protocol, there should be plenty of different ones published in various books (tc protocols vol 1-3 if i remember right) or try n get ahold of the book: plants from test tubes (a pretty straightforward "low level" intro book to TC), but the new edition should have more protocols added to it (incl. dicotyl plants species) hence, if you find a sterilization protocol for let's say tobacco or tomatoes, you should have no troubles to tweak that one to your own needs... or google for research/white papers on TC sterilization protocols.

most common agents used are: natrium hypochlorite (NaClO), ethanol/ethyl-alcohol (70%), and some very specialized compounds like nano-silver and nano titanium formulations (highly "experimental" also the nanoparticles n formulations could cause serious damage to the lab tech guy if proper safety meassures ain't undertaken -> stick to alc n NaClO)... ofcourse followed by single or multiple sterile aqua dest rinse(distillated water which has been autoclaved)...

blessss
ps.: good luck

 

[sonofsour]

I am unable to reveal data about compounds or processes , understandably the corporation that employs me would be less than happy if I was to map processes , I have put it on the line a bit to be able to share what I am so please understand that I am simply not able to offer any specifics , there does exist high concentrate compounds that are costly but will help you with your issues !
Sincerely
Charles

Hello Charles just wondering if your with a LP?

 

[xxxstr8edgexxx]

do you all know of any supply companies selling medium and hormone preloaded petris for induction of shoots and rooting.

 

[xxxstr8edgexxx]

the recipes from the ol miss studies are not going to travel well according to the lab that will preload petris that will ship i had forgotten about htis thread til i got that rep comment..

 

[Lyfespan]

Can I ask why the TC over cloning?

 

[xxxstr8edgexxx]

Can I ask why the TC over cloning?

its not an either or situation. traditional stem cutting ex vitro cloning will always be the way cannabis is propagated by clone for production.


1) for shelf space mom rooms holding breeder males numerous phenos and breeder parents to be brought out of petri to make new plants to repeat a breeding line or just to keep strains on ice with out having to manage a big grow area for all those moms. thousands of phenos could fit in a shelf system in a very very small area with minimal maintainance or power usage. extremely efficient.

2)pathogen control. most pathogens and especially bugs are not going to survive the process.
much easier to control an out break on a clone the size of a pencil eraser than a bush.

if you clone the new meristems cells you eliminate a lot of virus's from the host mother plant. the viruses typically dont infect the newly emerged meristem cells until they've been growing for a day or two. so in a lab setting under a scope you could extract these tiny cells as they emerge and clone them for a plant very similar to the plant as it existed when it came from seed.

3) ease of descreet transport across state and national borders via post.

4)mass propigation. if you need clones in the tens of thousands it becomes extremely efficient on a time scale. in thr hundreds or even thousands regular cloning methods would be the method. but for more, doubling every two weeks from tc woould get you there much faster at a certain point of scale.

 

[Lyfespan]

its not an either or situation. traditional stem cutting ex vitro cloning will always be the way cannabis is propagated by clone for production.


1) for shelf space mom rooms holding breeder males numerous phenos and breeder parents to be brought out of petri to make new plants to repeat a breeding line or just to keep strains on ice with out having to manage a big grow area for all those moms. thousands of phenos could fit in a shelf system in a very very small area with minimal maintainance or power usage. extremely efficient.

2)pathogen control. most pathogens and especially bugs are not going to survive the process.
much easier to control an out break on a clone the size of a pencil eraser than a bush.

if you clone the new meristems cells you eliminate a lot of virus's from the host mother plant. the viruses typically dont infect the newly emerged meristem cells until they've been growing for a day or two. so in a lab setting under a scope you could extract these tiny cells as they emerge and clone them for a plant very similar to the plant as it existed when it came from seed.

3) ease of descreet transport across state and national borders via post.

4)mass propigation. if you need clones in the tens of thousands it becomes extremely efficient on a time scale. in thr hundreds or even thousands regular cloning methods would be the method. but for more, doubling every two weeks from tc woould get you there much faster at a certain point of scale.

Thank you for the very detailed break down, I was just asked to look into this by a friend. Now I can understand why lol.

 

[wodi]

Very interesting

 

[xxxstr8edgexxx]

its a matter of being able to pass cloned genetics to another grower with the most likelyhood of not passing a pathogen or pest. magine being able to order garanteed genetics in a jar full of what would look like a jar full of frog eggs the size of gumballs that are each an exact clone garanteed clean as a seed. they would be much cheaper than cuttings too. far cheaper to produce. less space and much more hygienic.