Who is our resident Tissue culture master?

[englishrick]

A fair few people offer viroid testing,,,I was kinda into a company called delta leaf but ive heard bad reviews since ,,

I believe tests for about 8 different viroids are available nowadays ,,

Do you guys have any favourite companies at the moment?

 

[Rico Swazi]

I wasn't planning on putting pcr tests in the kit ,,I could do with understanding testing a lil more,

no tests? sorry to hear that. You are missing a huge opportunity
As I stated earlier, I see a definite need and huge market for inexpensive PCR testing kits for the home gardener.

In tissue culture ,we atempt to isolate the apical meristems because they are always clean,,,apical meristem tissue has not had chance to become infected,,older tissue becomes infected quickly and to avoid infected tissue we isolate the apical meristem,,,does this make sense to you now?

No. What you say doesn't make sense. You can say it a thousand times.
The paper I cited says different. Did you bother to read it?
how about the text in bold?

In addition, if meristem culture of cannabis is used to obtain pathogen-free plantlets, it would have to be accompanied by a similar PCR-based assay to test for the absence of these pathogens.

Thought you were further along with understanding PCR testing and protocol . Seems I was mistaken.
Good luck with your business venture and do consider adding RT-PCR testing when you can
Offering a choice of one or all of the available viroid tests would be a very good start
FWIW, I speak as a potential customer seeking a way to test for stable genetics

 

[englishrick]

no tests? sorry to hear that. You are missing a huge opportunity
As I stated earlier, I see a definite need and huge market for inexpensive PCR testing kits for the home gardener.



No. What you say doesn't make sense. You can say it a thousand times.
The paper I cited says different. Did you bother to read it?
how about the text in bold?


Thought you were further along with understanding PCR testing and protocol . Seems I was mistaken.
Good luck with your business venture and do consider adding RT-PCR testing when you can
Offering a choice of one or all of the available viroid tests would be a very good start
FWIW, I speak as a potential customer seeking a way to test for stable genetics

I'm not at a point where I can manufacture viroid tests,,,sorry bro

the apical meristem is a tiny tip at the top,,, are we talking about the apical meristem here?,,,il go check this paper you posted

 

[englishrick]

I think this is the part you are talking about



A polymerase chain reaction-based assay showed conclusively that cannabis stem tissues contained a range of fungi. The method allowed the detection of 1 ng/ml of genomic DNA and could be used to screen donor plants to determine the background level of microbial contamination. Similar PCR-based methods have been used to screen mother plants and tissue-cultured plants such as strawberries, sweet potatoes, and roses to ensure they are free of bacteria and fungi (Moreno-Vázquez et al., 2014; University of California Davis, 2008).



This is good practice,,yes,,contamination gets in most of the time,,testing is great,, if you can do loads of its even better,,it's not cheap tho,,







This approach can be applied to cannabis plants before they are deployed in tissue culture. In addition, if meristem culture of cannabis is used to obtain pathogen-free plantlets, it would have to be accompanied by a similar PCR-based assay to test for the absence of these pathogens.



hes talking about using apical meristems to obtain pathogen-free genetics,,yes of you will need to test ,,this is to check for contamination seeking through,,,it's not cheap and doesn't seem to be simple to manufacture





Nodal explant cultivation is unlikely to be free of pathogens given the high levels of internal contamination observed in this study. Therefore, shoots derived from nodal cultures should be avoided because of the potential for contaminants. Meristems represent the explant of choice to obtain pathogen-free plantlets from tissue cultures of C. sativa.





notice this last part I put in bold,,,yes testing is essential to be absolutely sure,,but not everyone can afford it at this level,,, I don't know enough about the reagents and bits to manufacture home kits,,,,most companies ask for samples to be sent into there lab,

since apical meristems start clean and become infected , apical meristems are the best choice for clean samples ,,the idea is to prevent contamination,,,we do this with initiation,,,my previous idea was an atempt to limit contamination via pre treatments,,heat treatments, uvc treatments, h2o2 treatments, anti fungal, ppm, antibiotics etc, ,my thoughts were at what point do the treatments negate the need for apical meristem or indirect organogenesis when it comes to removing pathogens, fungus, bacteria etc

 

[GMT]

Whole world is full of fungal spores, the second you take it out of the tube, it's infected again. It's more useful for bacterial issues I feel.

 

[Rico Swazi]

yeah, infected with ??? The science to determine such is fascinating as well as frustrating

 

[Rico Swazi]

I think this is the part you are talking about







This is good practice,,yes,,contamination gets in most of the time,,testing is great,, if you can do loads of its even better,,it's not cheap tho,,

thanks for reading Rick
yes , the very first sentence of that paragraph is what I was referring to

A polymerase chain reaction-based assay showed conclusively that cannabis stem tissues contained a range of fungi

I suggested it was not ' always clean' as you previously stated and now I hear a different tune?

This is good practice,,yes,,contamination gets in most of the time,,testing is great,, if you can do loads of its even better,,it's not cheap tho,,

Also, reverberating 'apical meristems are the best choice for clean samples ' while true in itself
does not imply pathogen free or validate your statements 'always clean' or 'starts clean'.
Statements I take issue with
Perhaps 'always cleaner' and ' starts cleaner' would be better terms though its your call in the future.
Gives a false sense of stability IMHO to say it in absolute terms like that...

I agree PCR is expensive enough not to be included at this time ,
have you or anyone tried any of the other less expensive testing methods such as lateral flow assay and/or ALISA ?

 

[GMT]

yeah, infected with ??? The science to determine such is fascinating as well as frustrating

Outside of a satellite lab, mold and fungus is in the air, you can't have anything in the real world that is free from contamination. The issue is always whether or not it reaches a level of importance.
Viruses are tricky. In theory, once a cell is infected, it only produces viruses, not more cells. Therefore in theory, the points at which new growth is being initiated, must be free of viruses.
The method of infection is paramount. This is why I would only suggest the root tips. Due to their protection from infection they offer a superior source of cells than anything above the ground could. The chances of an A.M. from above, being clean, depends more on the environment than the location in my view.

 

[englishrick]

thanks for reading Rick
yes , the very first sentence of that paragraph is what I was referring to

I suggested it was not ' always clean' as you previously stated and now I hear a different tune?

This is good practice,,yes,,contamination gets in most of the time,,testing is great,, if you can do loads of its even better,,it's not cheap tho,,

Also, reverberating 'apical meristems are the best choice for clean samples ' while true in itself
does not imply pathogen free or validate your statements 'always clean' or 'starts clean'.
Statements I take issue with
Perhaps 'always cleaner' and ' starts cleaner' would be better terms though its your call in the future.
Gives a false sense of stability IMHO to say it in absolute terms like that...

I agree PCR is expensive enough not to be included at this time ,
have you or anyone tried any of the other less expensive testing methods such as lateral flow assay and/or ALISA ?


View attachment 18809542

No tune change,,I kinda find that a lil offensive,,I think you are missing that I'm attempting to mediate the point at which apical meristem work is unnecessary for removal of viroids due to perfect initiation protocol,,,

in the world of hard science the best approach to clean genetics is to do the work at any cost, apical meristem is the job with flawless initiation,,I dont really work for the world of hard science so I'm trying to make it easy for the home grower

Anyway,,, lets get back to the paper,, should fungus be a concern ?,, fungus is the contaminant in question here,,why did they not use systemic fungal inhibitors???,, this is something that cost nothing and would probably have totally changed the outcomes,,reliable initiation is the big issue here

When we talk about infection, the apical meristem is not inherently infected ,,tissue becomes infected,,,,,gmt is correct,,in most situations shit becomes infected the second its out of invitro,,,

Hay Rico,,the problem is contamination,,with better initiation i believe we can avoid it,,,are you suggesting that the apical meristem is impossible to isolate or are you suggesting the contamination is systemic and nothingcan be done??,,No I've not tried any of the test you listed but I'm definitely interested,,thanks for listening them,,

 

[GMT]

I do think this is the right way to go. Baby steps. You will probably get 95% of the benefits of T.C. by following the protocols, with small clones, and saving 95% of the problems of T.C.
Clearly full T.C. is superior, but in what percentage of the situations, home growers are faced with?

 

[Rico Swazi]

no offense mate, saying the same thing you and GMT in that

,in most situations shit becomes infected the second its out of invitro,,,

I'll double down and say the apical meristem IS inherently infected as the paper states
and ...
going all in with the crazy notion somaclonal mutations are a result of such infections

agreeing to disagree is best thing we do unless you have something other than opinion rick

 

[GMT]

Rico, did you read that link I posted on root tips? Interesting read.

 

[englishrick]

no offense mate, saying the same thing you and GMT in that

I'll double down and say the apical meristem IS inherently infected as the paper states
and ...
going all in with the crazy notion somaclonal mutations are a result of such infections

agreeing to disagree is best thing we do unless you have something other than opinion rick

I am of the opinion that it is possible to scrubb every using apical meristem invitro,,but honestly I can't be bothered pushing the point so let's just say we disagree,,,The paper does say in conclusion that contamination is unavoidable, but i think initiation is lacking,,,

I like the idea of using root tips ,, not done it myself yet tho,,,Indirect organogenesis is pretty standard in tissue culture so its not hard

 

[englishrick]

no offense mate, saying the same thing you and GMT in that

I'll double down and say the apical meristem IS inherently infected as the paper states
and ...
going all in with the crazy notion somaclonal mutations are a result of such infections

agreeing to disagree is best thing we do unless you have something other than opinion rick

I kinda liked this post,,it brewed nicely

I'm intrigued about somaclonal variations being due to possible fungal interactions,,,it's not a bad thought at all,,,

To be fair,,that paper made me think about fungal interactions a lil more,,,are we considering them enough of a contamination to warrant more work?,,please correct me if im wrong, but the only contamination detected at the end of that paper was fungal???,,

I don't agree with the conclusions of that paper,, id like to have seen systemic fungal inhibitors being used and other treatments,,,imho that paper seems to be lacking in initiation,,,when I can be bothered il argue the toss,,at some point il just lay down my own initiation protocol and you can test it for yourself,,

Usaly, with genetic maintenance multiple samples are taken and backups save the day if somaclonal variations are detected ,,,I like the idea of giving a reason to the variations,,at the moment big science just sorta counts it as random transcription errors,,,you can induce it with frequency treatments but when we are looking to stop it we have previously had no answers

 

[Rico Swazi]

Rico, did you read that link I posted on root tips? Interesting read.

Busy busy, apologies for the late reply
Yes, old paper with not much supporting evidence based on cytoplasmic streaming so let it be.
more recent papers suggest root border cells ( slime cap exudates) and their defensive 'traps' can be weakened and overcome-

Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped.

https://europepmc.org/article/MED/27336156

search - extracellular DNases (exDNases) for more

I kinda liked this post,,it brewed nicely

I'm intrigued about somaclonal variations being due to possible fungal interactions,,,it's not a bad thought at all,,,

Rick, happy as hell the lateral transfer was successful
here is a bit more on somaclonal mutation/variation if you like
note 'all somatic tissue culture can result in somaclonal variation' including root tip

The term somaclonal variation by Larkin and Scowcroft (1981) was given for the variability generated by the use of a tissue culture cycle.
Somaclonal variation is defined as genetic variation observed among progeny plants obtained after somatic tissue culture in vitro.
Theoretically all progeny plants regenerated from somatic cells should be identical clones. However, variations might occur in number of progeny which are known as somaclones and they are genetically variable from their explant.
The initiating explant for a tissue culture cycle may come virtually from any plant organ or cell type including embryos, microspores, roots, leaves and protoplasts. So, all somatic tissue culture can result in somaclonal variation.
Somaclonal variation is a phenotypic changes as a result of chromosomal rearrangement during tissue culture.

Somaclonal variation: Basis, Applications and limitations Biotechnology

The term somaclonal variation by Larkin and Scowcroft (1981) was given for the variability generated by the use of a tissue culture cycle.Somaclonal variation is defined as genetic variation observed among progeny plants obtained after somatic tissue culture in vitro.Theoretically all progeny...

www.onlinebiologynotes.com

7. Transposable elements has been especially interesting to me

,at some point il just lay down my own initiation protocol and you can test it for yourself,,

A cleaning/ disinfecting protocol and kit for explants during the initiation stage is long overdue
time to put yer skates on, chop chop and finish what you started
luck on all fronts mate

 

[acespicoli]

Picked up this book along with a few others probably 2 years ago
I think the biggest thing was lack of equipment,

There was this show back in the day

MacGyver (1985 TV series) - Wikipedia

en.wikipedia.org

Any way making due with what you do have is huge, forget a large investment
Unless of course its for profit
For me its a small scale hobby so alot of diy gear
Im glad to find this thread im along for the show

Keep it sterile with

Cleanroom - Wikipedia

en.wikipedia.org

This seemed like one of the most versatile recipes although there are extreme simple recipes

Murashige and Skoog medium - Wikipedia

en.wikipedia.org

This makes me wonder what is the most micro clone which could be mailed???
Hope to learn some new things specific to canna biology!

 

[Loc Dog]

Has anyone found a method they like?? Saw a youtube video but they were not concerned with with viroids or mold in the plant. It was basic flow hood, sterilize everything and put pieces (a couple of inch) in agar mixed with nutrients.

 

[englishrick]

Yeh of course,,,the part you are talking about is surface sterilisation,,,it's part of the tc process but it's not the part that avoids the viroids,,,to scrub viroids you will need to do something called apical meristem culture,,,it's basically a stage ahead of surface sterilisation

There are loads of mixes,,,,I have my own personal mix that I keep private, but most MS mixes will do the job,,,I add magic bits like plant preservative mixture etc,,when you start reading and testing, then you will make your own mix and improve on the standard hemp MS mix, once you do "step one (surface sterilisation) or initiation as its called in the books,,then you can step forward to higher level practical work like calus culture (indirect organogenesis) ,,apical meristem culture, anther culture, somatic fusion,,, enzymatic cell wall stripping etc,,it's comes in levels and modules

 

[Dr.Mantis]

Yeh of course,,,the part you are talking about is surface sterilisation,,,it's part of the tc process but it's not the part that avoids the viroids,,,to scrub viroids you will need to do something called apical meristem culture,,,it's basically a stage ahead of surface sterilisation

There are loads of mixes,,,,I have my own personal mix that I keep private, but most MS mixes will do the job,,,I add magic bits like plant preservative mixture etc,,when you start reading and testing, then you will make your own mix and improve on the standard hemp MS mix, once you do "step one (surface sterilisation) or initiation as its called in the books,,then you can step forward to higher level practical work like calus culture (indirect organogenesis) ,,apical meristem culture, anther culture, somatic fusion,,, enzymatic cell wall stripping etc,,it's comes in levels and modules

Hey englishrick,

Just wanted to say thank you. I’m getting geared up to explore inducing polyploidy in cannabis clones, and to me some level of TC will likely be needed. This thread, and your info has been the kick in the pants I need to get serious about it. I’ll be setting my space up in a 5x3 tent. Have you explored DKW media? From some recent lit I have seen, it looks to be a little more friendly to cannabis. I prepared the basal salts and vitamins for 100l of media, since I had the reagents lying around. There are also some interesting additives I’ve seen mentioned, like silicates, active carbon, and some transition metals.

Thanks again!

 

[englishrick]

I'm really happy to hear you say that,,,im all about helping individuals make some progress ,,,when it comes to personal people doing personal research i can't support you enough,,

DKW displays improved callogenesis compared with MS basal salts in some cultivars

What have you read so far?