Cannabis grafting

Darpa · Aug 16, 2019

F2F said:
This thread is fascinating for someone who has plant number problems/mother keeping limitations like me.

I've been really interested in figuring out how to maintain genetics with minimal upkeep. Clone storage in the fridge works and is a great asset.

Can one prepare a scion and keep it in suspended animation for some period of time, to graft at a later date?!

Cheers!
F2F

Hi F2F,
I've grafted scions that were about one month old (after they were cut from the mother plant. I Simply keep them in a a masson jar with about one inch of water at the Bottom. I changed the water every Week or so, and they were Under indirect fluorescent light).

This fall, I'll will perform an experiment to see how long I could preserve freshly cut scion with different preservation methods. We will see If I could preserve them for up to six month with low maintenance technique. I've already studied some technique with other plant species, so we'll how it turn out with cannabis.

I'll document everything here!

Cheer

Darpa

CroptimusPrime · Aug 16, 2019

This could be an interesting Fall. Imagine the huge number of diverse specimens one could collect this way. Do scions count as individual plants for plant-count purposes?

F2F · Aug 16, 2019

"Cellared" scions FTW!

"Cellared" scions FTW!

Sam and Darpa :thank you:

Things will get interesting next year with this technique paired with OD!

Wonderful knowledge and learnings here.

Darpa - let us know how that long term experiment goes!

All the best,
F2F

AgentPothead · Aug 16, 2019

CroptimusPrime said:
This could be an interesting Fall. Imagine the huge number of diverse specimens one could collect this way. Do scions count as individual plants for plant-count purposes?

I'm pretty sure they are counting the physicalness of the plant, the roots the stems the leaves. The genetics shouldn't matter right? Even if you had 20 strains on 1 rootbase, it's still just 1 plant isn't it?

Darpa · Aug 17, 2019

AgentPothead said:
I'm pretty sure they are counting the physicalness of the plant, the roots the stems the leaves. The genetics shouldn't matter right? Even if you had 20 strains on 1 rootbase, it's still just 1 plant isn't it?

Yes! The plant count is based on the number of rootstock growth. You could have dozen of strains grafted of a single rootstock, Under the law, it a single plant…

My interest in grafting came partially from legal plant number limitation...

Darpa

Hookahhead · Aug 17, 2019

Based on the previous posts, I think he was referring to scions in a bag in the fridge for storage?

Hopefully these would be considered “plant material” the same as leaves, stems, etc. instead of actual plants. However, I do not know the real legality on this, and it may vary by location.

Darpa · Aug 17, 2019

Hi IC friends,

That °Cold Storage idea° is realy appeling to me, and Im willing to do an experiment if some of you are interested in the concept. If I get 5 peoples that found this post helpfull or that want to see this experiment, here is what i'm planning to do:

EXPERIMENT ON COLD STORAGE OF CANNABIS TISSUS FOR GRAFTING PURPOSE.

Here is a summary of the proposed protocol:

4 cuts of my (((Biker kush x (GG4 x (GSC x Arcata TrainWreck))) speciment will be selected for cloning next week. These will be use as Root Stock.

About 30 cuts of differents cultivars will be selected. They will be about 3 to 4 inch long with 4 to 4 auxillary buds site on each cuts.

They will undergo quick wash sterilisation with hypochlorite solution since fungus and mold contamination will be the biggest issu of this experiment…

I'm intending to graft cold preserved scions (under different preservation condition) at a monthly interval with the micro auxilary buds technique (see post 161) .

So in other word, all of the scions will be recolted at the same time, and graft will be performed at 1 month, 2 month, 3 month (you guys get the principle, up to one year) of cold preservation.

Here is the cold preservation technique I'm planning to test:

4 degree in breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media (24h of light, low) (The type of bags
used to grow mushroom mycelium in aseptic technique)

4 degree in breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media (darkness)

4 degree in steril beaker covered with breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media (darkness)

4 degree in steril beaker covered with breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media (24h of light, low)

Then, to spice up the experiment, I'll try -20 Celcius preservation also. Cuts will be placed in steril beaker covered with breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media.

The preservation media will be non freeze complex cryoprotectans composed of:

One sample of: 10% DMSO, 10% propylene glycol，10% PEG，10% glycerin) to avoid ice crystal formation when tissue stored in -20℃

One sample of: 10% propylene glycol，10% PEG，10% glycerin and sucrose)

All the material and media will undergo autoclave sterilisation.

Anyone interested? Please feel free to comment the protocol. All the suggestion are welcome!

Cheer

Darpa

Sam_Skunkman · Aug 17, 2019

You will need to pretreat the materials to be frozen with something to prevent the formation of ice crystals in the plant. I tried freezing both callus and meristems and they both died when thawed out, they turned into black goo and died and could not be regrown, I did this 25 years ago. Today there are protocols to pretreat the plant materials with treatment like DMSO & sugars, that prevent the freezing from causing ice formation inside the plant materials.
Here is a paper from this year:

https://sci-hub.tw/10.1159/000496869
Cryopreservation of Shoot Tips of Elite Cultivars of Cannabis sativa L. by Droplet Vitrification
Esther Uchendu, Hemant Lata, Suman Chandra, Ikhlas A. Khan, Mahmoud A. ElSohly
Med Cannabis Cannabinoids
Accepted: January 8, 2019
DOI: 10.1159/000496869
Cannabis sativa L. (marijuana or hemp) is recognized worldwide for its psychoactive properties as well as for fiber production. This study focused on the evaluation of 3 droplet vitrification protocols for long-term conservation of shoot tips in liquid nitrogen (LN). Shoot tips (∼0.5 mm) were excised from 3- to 4-week-old in vitro-grown shoots of 3 cultivars (MX, VI-20, and B-5: high tetrahydrocannabinol [THC], high cannabidiol [CBD], and intermediate THC∼CBD, respectively) and pretreated on 5% dimethyl sulfoxide agar plates
for 48 h. The shoot tips were then vitrified in LN using 3 separate cryoprotectant (plant vitrification solutions [PVS] #2, #3, and #4) droplets on an aluminum cryoplate. There was no significant difference between the regrowth of cryopreserved shoot tips exposed to PVS2 for 15 and 20 min, but
regrowth of all 3 cultivars significantly declined after 20 min of exposure. Exposure duration of 15 min was adapted for subsequent experiments. Regrowth of cryopreserved MX was significantly higher with PVS2 (63%) than with PVS3 and PVS4 (≤5%). Regrowth of cryopreserved VI-20 was highest with PVS2 (57%) and significantly higher than with PVS3 and
PVS4 (≤25%). The regrowth of cryopreserved shoot tips of B-5 was significantly different between all 3 protocols with PVS2 > PVS4 > PVS3. Both PVS2 and PVS4 produced regrowth above 55%, while regrowth with PVS3 was significantly lower (31%). These results indicate that 15–20 min of exposure to PVS2 are most suitable for cryopreservation of these varieties.
This is the first report on protocol development for the cryopreservation of organized tissues of C. sativa L. for germplasm conservation

-SamS

Darpa said:
Hi IC friends,

That °Cold Storage idea° is realy appeling to me, and Im willing to do an experiment if some of you are interested in the concept. If I get 5 peoples that found this post helpfull or that want to see this experiment, here is what i'm planning to do:

EXPERIMENT ON COLD STORAGE OF CANNABIS TISSUS FOR GRAFTING PURPOSE.

Here is a summary of the proposed protocol:

4 cuts of my (((Biker kush x (GG4 x (GSC x Arcata TrainWreck))) speciment will be selected for cloning next week. These will be use as Root Stock.

About 30 cuts of differents cultivars will be selected. They will be about 3 to 4 inch long with 4 to 4 auxillary buds site on each cuts.

They will undergo quick wash sterilisation with hypochlorite solution since fungus and mold contamination will be the biggest issu of this experiment…

I'm intending to graft cold preserved scions (under different preservation condition) at a monthly interval with the micro auxilary buds technique (see post 161) .

[URL=https://www.icmag.com/ic/picture.php?albumid=70944&pictureid=1689875&thumb=1]View Image[/url]

So in other word, all of the scions will be recolted at the same time, and graft will be performed at 1 month, 2 month, 3 month (you guys get the principle, up to one year) of cold preservation.

Here is the cold preservation technique I'm planning to test:

4 degree in breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media (24h of light, low) (The type of bags
used to grow mushroom mycelium in aseptic technique)

4 degree in breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media (darkness)

4 degree in steril beaker covered with breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media (darkness)

4 degree in steril beaker covered with breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media (24h of light, low)

Then, to spice up the experiment, I'll try -20 Celcius preservation also. Cuts will be placed in steril beaker covered with breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media.

The preservation media will be non freeze complex cryoprotectans composed of:

One sample of: 10% DMSO, 10% propylene glycol，10% PEG，10% glycerin) to avoid ice crystal formation when tissue stored in -20℃

One sample of: 10% propylene glycol，10% PEG，10% glycerin and sucrose)

All the material and media will undergo autoclave sterilisation.

Anyone interested? Please feel free to comment the protocol. All the suggestion are welcome!

Cheer

Darpa

Tony21 · Aug 17, 2019

Darpa said:
Hi IC friends,

That °Cold Storage idea° is realy appeling to me, and Im willing to do an experiment if some of you are interested in the concept. If I get 5 peoples that found this post helpfull or that want to see this experiment, here is what i'm planning to do:

EXPERIMENT ON COLD STORAGE OF CANNABIS TISSUS FOR GRAFTING PURPOSE.

Here is a summary of the proposed protocol:

4 cuts of my (((Biker kush x (GG4 x (GSC x Arcata TrainWreck))) speciment will be selected for cloning next week. These will be use as Root Stock.

About 30 cuts of differents cultivars will be selected. They will be about 3 to 4 inch long with 4 to 4 auxillary buds site on each cuts.

They will undergo quick wash sterilisation with hypochlorite solution since fungus and mold contamination will be the biggest issu of this experiment…

I'm intending to graft cold preserved scions (under different preservation condition) at a monthly interval with the micro auxilary buds technique (see post 161) .

https://www.icmag.com/ic/picture.php?albumid=70944&pictureid=1689875 View Image

So in other word, all of the scions will be recolted at the same time, and graft will be performed at 1 month, 2 month, 3 month (you guys get the principle, up to one year) of cold preservation.

Here is the cold preservation technique I'm planning to test:

4 degree in breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media (24h of light, low) (The type of bags
used to grow mushroom mycelium in aseptic technique)

4 degree in breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media (darkness)

4 degree in steril beaker covered with breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media (darkness)

4 degree in steril beaker covered with breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media (24h of light, low)

Then, to spice up the experiment, I'll try -20 Celcius preservation also. Cuts will be placed in steril beaker covered with breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media.

The preservation media will be non freeze complex cryoprotectans composed of:

One sample of: 10% DMSO, 10% propylene glycol，10% PEG，10% glycerin) to avoid ice crystal formation when tissue stored in -20℃

One sample of: 10% propylene glycol，10% PEG，10% glycerin and sucrose)

All the material and media will undergo autoclave sterilisation.

Anyone interested? Please feel free to comment the protocol. All the suggestion are welcome!

Cheer

Darpa

Very interested, very cool.

AgentPothead · Aug 17, 2019

Hookahhead said:
Based on the previous posts, I think he was referring to scions in a bag in the fridge for storage?

Hopefully these would be considered “plant material” the same as leaves, stems, etc. instead of actual plants. However, I do not know the real legality on this, and it may vary by location.

Duh, sorry, I didn't know what a scion was so don't mind me. :biggrin:

F2F · Aug 17, 2019

Interested here also DARPA!

Darpa said:
Hi IC friends,

That °Cold Storage idea° is realy appeling to me, and Im willing to do an experiment if some of you are interested in the concept. If I get 5 peoples that found this post helpfull or that want to see this experiment, here is what i'm planning to do:

EXPERIMENT ON COLD STORAGE OF CANNABIS TISSUS FOR GRAFTING PURPOSE.

Here is a summary of the proposed protocol:

4 cuts of my (((Biker kush x (GG4 x (GSC x Arcata TrainWreck))) speciment will be selected for cloning next week. These will be use as Root Stock.

About 30 cuts of differents cultivars will be selected. They will be about 3 to 4 inch long with 4 to 4 auxillary buds site on each cuts.

They will undergo quick wash sterilisation with hypochlorite solution since fungus and mold contamination will be the biggest issu of this experiment…

I'm intending to graft cold preserved scions (under different preservation condition) at a monthly interval with the micro auxilary buds technique (see post 161) .

[URL=https://www.icmag.com/ic/picture.php?albumid=70944&pictureid=1689875&thumb=1]View Image[/url]

So in other word, all of the scions will be recolted at the same time, and graft will be performed at 1 month, 2 month, 3 month (you guys get the principle, up to one year) of cold preservation.

Here is the cold preservation technique I'm planning to test:

4 degree in breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media (24h of light, low) (The type of bags
used to grow mushroom mycelium in aseptic technique)

4 degree in breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media (darkness)

4 degree in steril beaker covered with breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media (darkness)

4 degree in steril beaker covered with breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media (24h of light, low)

Then, to spice up the experiment, I'll try -20 Celcius preservation also. Cuts will be placed in steril beaker covered with breathable steryle bag with 0.2 Micron Filter for air exchange with humidity media.

The preservation media will be non freeze complex cryoprotectans composed of:

One sample of: 10% DMSO, 10% propylene glycol，10% PEG，10% glycerin) to avoid ice crystal formation when tissue stored in -20℃

One sample of: 10% propylene glycol，10% PEG，10% glycerin and sucrose)

All the material and media will undergo autoclave sterilisation.

Anyone interested? Please feel free to comment the protocol. All the suggestion are welcome!

Cheer

Darpa

bsgospel · Aug 17, 2019

I'm interested. Right on, Darpa.

CroptimusPrime · Aug 17, 2019

I'm also interested, Darpa, if you're still counting. Also, what about alcohol to lower the freezing/crystallization point?

Darpa · Aug 19, 2019

F2F said:
Sam and Darpa

Things will get interesting next year with this technique paired with OD!

Wonderful knowledge and learnings here.

Darpa - let us know how that long term experiment goes!

All the best,
F2F

Here is an update on the Multi Strain Graft Experiment.

Plants are over 7 feets and female inflorescence are showing up. The flowering stretch didn't realy start yet.

Also, performing Monster Cropping gave me so many branchs to work with at the begining. And then Super Cropping the grafted scion resulted in a realy bushy beast….

Cheer

Darpa

Darpa · Aug 19, 2019

Monster cropping & Super cropping effect:

From:

To:

Darpa

CroptimusPrime · Aug 19, 2019

Wow. These two techniques seem like they're even more beneficial to as addenda to the techniques of grafting itself than they are to ordinary growing. Essential, even.

Darpa · Aug 20, 2019

Hi IC friend,

For those of you who are concerned about the weekness-fragility of a graft (like me at the begining of the experiment) after just few weeks, the fusion zone is not visible anymore. (see post 252 and 263)

I was first affraid that those area would break under strong stromy winds but the fusions zones is almost indistinguishable from the root stock steams... The red tape is there only because the initial labeling was washed out from the rain…

At the end of the season, I'll make longitudinal cuts of the fusion zone and take microscopic picture of the healing... That would be interesting...

At the end it all about big yeild, multi strain and legal plants count...

Cheer

Darpa

Darpa · Aug 20, 2019

Also,
I wondering, why waste such a nice root stock that when from Monster Cropping and Super Crooping.

At the end of the season, I'll cut all of the main branch and keep the root stock with some of it's racinal system and try to graft scion on the branch. I did experiment that last year but I got mold contamination before I was able to try grafting.

This year, I'll trasfert the root stock in a grow tent, no fan to keep huminidy high, and trim all the big roots like I use to do when I was growing Bonsai. Media will be composed of HP pro mix with 50% perlite....

That would give me at least 25 major shoots to work with...

Darpa

FrostAie · Aug 20, 2019

With longer autos around 100 day ones people have had success with shorter cycle autos i would watch out

CroptimusPrime · Aug 21, 2019

FrostAie said:
With longer autos around 100 day ones people have had success with shorter cycle autos i would watch out

Pardon my density, but what would the negative outcome be of applying any of this to a <100-day auto?

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