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Who is our resident Tissue culture master?

englishrick

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The apical meristem of a plant is always clean,,,the idea here is to prevent it from becoming infected with treatment and prior surface cleaning, we then isolate with an explant,,,dumping the original plant stock,,
 

Mudballs2.0

Active member
Wouldn't a 70°C treatment for one day destroy the living plant sample?
it's weird..double strand DNA can withstand it, single strand RNA cannot. What im seeing inline with englishricks idea, is just short treatments...like 1 hr. Idk i. Just now trying to get up to speed with him since last night lol
 

Loc Dog

Hobbies include "drinkin', smokin' weed, and all k
Veteran
Yes growing mushrooms is verry much just growing cell masses and splitting them up. Then dont split and spawn to substrate when you want to make fruit body.

Fuck man a 50% failure rate doesn't seem so great. It was $35USD for a litters worth of pre mixed cannabis media. Thats like 20 containers - $1.75 a jar
I have to do more reading but im gonna start this as soon as i have a handle. I'll start with 10-fill a bunch of jars with loose lids and pressure cook em. Tighten the lids tomorrow in front of the hood and store em in the fridge untill i'm ready to move on.

I have to figure out how to sterilize the plant tissue. Also have to find my forceps...
Would Physan 20 work to sterilize plant material?
 

Loc Dog

Hobbies include "drinkin', smokin' weed, and all k
Veteran
The apical meristem of a plant is always clean,,,the idea here is to prevent it from becoming infected with treatment and prior surface cleaning, we then isolate with an explant,,,dumping the original plant stock,,
Do you know how many inches is always clean, and is that just the main stem top growing point or side branching also.
 

Mudballs2.0

Active member
what he's discussing is apical meristem, this is a root pic but it works the same to represent the growing branch tips.
Screenshot (13).png

Do you know how many inches is always clean, and is that just the main stem top growing point or side branching also.
not far at all, they literally shave that tip like shaving an onion for the skillet.
 

GMT

The Tri Guy
Veteran
I'd be interested to know if T.C. could actually be performed where you used root tips rather than the top 1-3 mm of plant growth site.
Root tips would offer more protection from infection than anywhere else.

 

NEED 4 SEED

Well-known member
I'd be interested to know if T.C. could actually be performed where you used root tips rather than the top 1-3 mm of plant growth site.
Root tips would offer more protection from infection than anywhere else.

From what I've read roots or tips should work the same
 
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Rico Swazi

Active member
Still processing this paper on bacterial/fungal endophytes
I see a definite need and huge market for inexpensive PCR testing kits for the home gardener.



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 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. 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.

 

NEED 4 SEED

Well-known member
As they said, the critical point are protocols for cannabis tissue culture. Where can we find them?
 

englishrick

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When I get chance il put the kit out,,,I promise,,,il explain everything and give you all the bits needed

Roots also have totipotency,,so yes roots can grow into a full plant,,,roots need to go through indirect organogenesis,,,meaning it will turn to callus (undifferentiated cells) and then through differentiation it will grow shoots or roots depending on the mediums ratios of auxin to cytokinins
 

englishrick

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what he's discussing is apical meristem, this is a root pic but it works the same to represent the growing branch tips.
View attachment 18808500
Do you know how many inches is always clean, and is that just the main stem top growing point or side branching also.
not far at all, they literally shave that tip like shaving an onion for the skillet.
You are absolutely correct,,thanks for the images,,I probably wouldn't bother myself so It helps alot ,,


When we talk about meristem we talk about normal cloning,,big ones,,,even microcloning is basically just normal meristem cloning, same as big clones,,,but when we talk about the apical meristem,,magic worlds being APICAL MERISTEM,, we talk about a tip so small you need a 50x+ stereoscopic microscope to do catch it,, notice one in the picture of my kit,,,


The apical meristem is Always clean,,the idea with the deeper end of tissue culture is to isolate and culture it,,
 

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Mudballs2.0

Active member
When I get chance il put the kit out,,,I promise,,,il explain everything and give you all the bits needed

Roots also have totipotency,,so yes roots can grow into a full plant,,,roots need to go through indirect organogenesis,,,meaning it will turn to callus (undifferentiated cells) and then through differentiation it will grow shoots or roots depending on the mediums ratios of auxin to cytokinins
Some of us got to talking about that heat treatment concept and i think we have one fella that's possibly gonna try it. He decided to dip infected clones in 158degree hot water over a series of days and then if it lives, send in for testing.
I want to thank you for being a sharing individual.
 

Mudballs2.0

Active member
Not for this particular RNA
Screenshot_20230203-142734_Samsung Notes.jpg

I guess we could extrapolate and correlate...bro, i hate math..much less calculus
 

grod31

Well-known member
Veteran
Great conversation guys, great links and a wealth of information.
My first TC jars are in the PC right meow. The directions say 20 mins but doesn't say at what PSI so I just went to 15. I hope 40 mins is okay cause I have other stuff in there that needs that long. I only used half the media incase I fucked it all up.
 

Rico Swazi

Active member
When I get chance il put the kit out,,,I promise,,,il explain everything and give you all the bits needed

Will that kit include RT-PCR?

'The apical meristem is Always clean' statement of yours has me confused
is this before or after initiation?
How could you know degree of cleanliness without a test is what I'm asking
 

englishrick

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

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?

Initiation is the process of bringing genetics into a tissue culture environment like invitro (in glass) testubes etc,,,,,initiation can start in the growroom prior to surface sterilisation, its this prior initiation work i was referring to in my previous posts,,

we take precautions like initiation, because of potential slips,,,we take precautions at all levels to prevent the possibility that when cutting tissue we might not avoid primordia or might not shave something enough,,small slips will fuk everything up, but if you take precautions at all levels then slight mistakes won't come back to haunt you,,,sometimes you can take the work too far,,,my previous post try to mediate what is entirely necessary
 
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GMT

The Tri Guy
Veteran
Unfortunately I suspect you'd need to know what you were testing for. Each undesirable would have its own markers making a universal test difficult if not impossible.
Prior samples could be sent for analysis and then PCR kits ordered for that issue I guess, if you live somewhere legal. Costly though.
 
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