Micropropagation - Plant Tissue Culture

[electro gypsy]

I know this has been around for several years now, but I'm not finding a lot of detailed information here on the topic. If there is please feel free to show me. I'd also like to hear from anyone who is practicing this currently. I gave it a brief try a few years ago with some success. Never made it to actual leaf producing plants, but it was so close it was promising. So I figured I would give it another go. Any input would certainly be appreciated!

Much love

 

[electro gypsy]

..and another go we shall have!

This is experiment one of what may be several. Depends on how fast we have success. I'll try and keep it as simple as possible. I am somewhat comfortable with this type of work in regards to fungi, so if I start rambling off terms or some gibberish that no one understands just let me know lol. This is going to be a live journal and I will be sort of piecing it together as we go.

Recipe #1

250ml / 1 cup well water warm
5 grams Agar Agar (Agar flakes)
2 teaspoons common processed cane sugar
1cc Canna Start
.5cc Superthrive
.5cc Canna Rhizotonic
50mg Thiamin
200mg Folic Acid
15mg Ba 6 Benzylaminopurine (Plant Hormone Cytokine)
15mg Ga3 Gibberellic Acid (Plant Hormone)
15mg IBA 3 Auxin / Indole Butyric Acid (Plant Root Hormone)


Tools needed at this point:

Measuring cup
Syringe (3cc preferable)
1) Pint canning jar
5) 1/2 pint canning jars (wide mouth)
PH meter
Scale
Pressure cooker

Directions:

Measure 250ml / 1 cup warm water and leave in cup. Add all ingredients except agar into the cup and mix until dissolved. Stir until well mixed. There may be some sediments but that's ok. You will see this though out most of the preparation. Now pour the mix into the 1 pint canning jar. Add the agar flakes and put the lid on tight. Rubber seal down.

Shake for a few minutes to mix and pour back into the measuring cup. Check the PH level. This one is done at 6.8 as it seems to be the most agreed upon level out of all the research I have done. Pour the mixture into the 1/2 pint jars dividing equally at approximately 50ml per jar. Wipe off any drops that may have occurred around the lip of the jar and threads. Put the lids on rubber seal up so that the metal side meets the glass. Tighten the lid ring until it won't turn and then loosen it approximately a quarter turn. You can now place the jars into your pressure cooker for sterilization.

Remember.. NEVER PUT TIGHT LIDS ON JARS INTO A PRESSURE COOKER. Unless you like cleaning up a sticky mess lol. Pressure cook at 15psi for 20 minutes. Leave the culture jars in the pressure cooker over night too cool.

..and this is exactly where we are right now.

 

[Weaselcracker]

I never heard of it till I heard you mention it. And I have no idea what it is or what you're about to do.
No don't tell me you'll ruin the surprise.
So here we are...

 

[electro gypsy]

A quick update. The cooled cultures came out of the pressure cooker this morning. The lids were tightened and everything is on the kitchen counter because I forgot to put it in the fridge this morning on my way out. Nothing to worry about here. They can be stored at room temps for a very long time. Provided they stay sealed and free of contamination.

..and there they sit.

 

[Big Sur]

Cool cool cool... I am interested in MJ TC. I have done it with orchids, but that was in a pro nursery and they had the media all set up in a lab environment.

Hmm, reminds me I need to water my baby clones today. Old school propagation by rooting cuttings.

 

[electro gypsy]

Cool cool cool... I am interested in MJ TC. I have done it with orchids, but that was in a pro nursery and they had the media all set up in a lab environment.

Hmm, reminds me I need to water my baby clones today. Old school propagation by rooting cuttings.

Howdy BS! Glad you could make it! Yep, I am of the old skool sort myself.. I highly doubt this will be the new way of cloning lol.. but it does fit some nice niches. My primary interest is in culture storage. I can also see some applications in last minute saves and tough to clone pheno's. I'm also interested in how it may apply to old seed revival and synthetic seeds. We have a lot of work ahead us my friend!

 

[electro gypsy]

OH! and that is exactly why I am doing this.. WE are doing this. To dissolve some of myth's and misconceptions. Home culture work is very possible, really not that hard and does not require a lab. The most expensive piece of equipment will likely be the pressure cooker.

Much love!

 

[Big Sur]

I'm also interested in how it may apply to old seed revival and synthetic seeds.

Had not thought of that, but I suppose you could carefully hull a seed and put it on TC growing medium. As long as the inside of the seed is white and not infected or dead.

Seed-assist! Now available online! Buy it now from MJ-Clone-R-Us!

 

[electro gypsy]

a side note regarding growth regulators cytokinins and auxins. stolen from the intertron..

Cytokinins in the intact plant are the phytohormones that work from the bottom up. Made in the roots (also seeds and fruits), cytokinins travel up the xylem and promote lateral growth. Since auxins travel down from the growing tip and act to suppress lateral growth, these two types of hormones strike a balance. This relationship persists in tissue culture. When the active concentration of the two hormones are balanced, callus formation and growth is favored. When cytokinin predominates, shoots are formed. Although the name “cytokinin” indicates that these molecules promote cell division, in reality cell division requires both hormones. Optimal hormonal conditions for propagation differ from species to species, and also differ with the stage of development

 

[electro gypsy]

another side note.. Dankly instigated research.

“Willow Water” – How it Works

“Willow Water” is a homebrew plant rooting hormone that is easily made and can be used to increase the strike rate (growth of roots) of cuttings that you’re trying to propagate.

The way that it works can be attributed to two substances that can be found within the Salix (Willow) species, namely, indolebutyric acid (IBA) and Salicylic acid (SA).

Indolebutyric acid (IBA) is a plant hormone that stimulates root growth. It is present in high concentrations in the growing tips of willow branches. By using the actively growing parts of a willow branch, cutting them, and soaking them in water, you can get significant quantities of IBA to leach out into the water.

Salicylic acid (SA) (which is a chemical similar to the headache medicine Aspirin) is a plant hormone which is involved in signalling a plant’s defences, it is involved in the process of “systemic acquired resistance” (SAR) – where an attack on one part of the plant induces a resistance response to pathogens (triggers the plant’s internal defences) in other parts of the plant. It can also trigger a defence response in nearby plants by converting the salicylic acid into a volatile chemical form.

When you make willow water, both salicylic acid and IBA leach into the water, and both have a beneficial effect when used for the propagation of cuttings. One of the biggest threats to newly propagated cuttings is infection by bacteria and fungi. Salicylic acid helps plants to fight off infection, and can thus give cuttings a better chance of survival. Plants, when attacked by infectious agents, often do not produce salicylic acid quickly enough to defend themselves, so providing the acid in water can be particularly beneficial.


Willow water can be made from cuttings of any tree or shrub of the willow family, a group of plants with the scientific name of Salix. The more cuttings that are used and the longer they are soaked in water, the stronger will be the resulting willow water. Recommendations for the exact method of soaking vary. Cold water can be used, and soaking times of four or more weeks are often quoted. Other gardeners use boiling water to steep the willow twigs and soak the mixture for around 24 hours.

 

[electro gypsy]

internal source

Homemade Rooting Hormone Willow Tea

 

[electro gypsy]

Quick update on the culture jars. Still sitting there lol. I forgot to mention that I placed the jars into ziploc sandwich baggies as an added protective barrier for the refrigerator. They will not be going there however. This weekend we will make a quick glove box aka still air box (SAB) and try dropping some plant tissue in there.

 

[electro gypsy]

Home now, smokin' Cenex and the Kratom is kicking in. Almost good to go lol.

Here's an example off an easy home made Still Air Box (SAB).

I'm not going to post the directions simply because there are so many variations. This can even be a cardboard box with clear plastic lol. All kinds of corners can be cut when designing this. I was actually thinking of making one real quick using a fish tank on its side with plastic sheeting and duct tape. I have a plastic tub but I would like to take pics while working possibly. So the glass would be nice.
Anyways, the objective is to create a mostly sealed environment that can be cleaned with bleach or alcohol spray. Disinfectants like Lysol can be used, but it has a history of causing a cellular disorder in fungi known as rosecomb. I have no idea how it will affect plant tissue and we are not even going there lol. I'll make the box tomorrow and then we can do some culture work.
Normally I would break out the big gun for this sort of work, but I want to show everyone just how easy home culturing can be when you get used to it.
This is a back pressured clean room with 2'x4' Hepa filter laminar flow hood. Not your everyday home appliance
And not something I get to show off very often lol. So I had to sneak it in here somehow. :icon_roll
Honestly, though a bit cumbersome, an SAB can be every bit as effective.

Much love!

 

[electro gypsy]

Managed to get the SAB built today. Fish tank on it side. 3/16" foam poster board for the entry points, and duct tape lol. Once everything is in and ready for work, the entry door will be sealed with tape and the hand holes will be slit so I can get my hands in there. I won't be using gloves for this box, just very clean hands and arms.
View from working side..

Sealed hand holes from inside..

Side view. Some stuff already in there, but I doubt I have time to culture tonight.

 

[Big Sur]

Looking good!

Where do you get 99% alcohol? The best I could find here was 91% when I was making hash oil one year.

 

[electro gypsy]

Looking good!

Where do you get 99% alcohol? The best I could find here was 91% when I was making hash oil one year.

it can be tough to find. the only two major stores i can find it at are Fred Meyer and Safeway. 99% is overkill for this but it's all i had.. for oil lol

 

[Big Sur]

it can be tough to find. the only two major stores i can find it at are Fred Meyer and Safeway. 99% is overkill for this but it's all i had.. for oil lol

Hmmmm, I have never seen it at Safeway or Freddies. But then, I was not looking for it there. I looked at places like Wal-whatever, CVS and Bi-Mart. I want to find some because the new law in Oregon allows me to make homemade hash using alcohol, and I can make up to and keep a POUND of it legally! With a half pound of bud plus a pound of hash? I should be able to stay high!

 

[electro gypsy]

Things have kind of gotten away from me these last few days. Very busy. Sorry for the delay. In the meantime I have been sneaking in a little reading at work lol. Taken from a pdf regarding synthetic seeds.

Plant material
Explants of nodal segements containing axillary buds,
were excised from multiple shoot cultures of high yielding
C. sativa variety (MX-1) maintained on Murashige and
Skoog's medium (Murashige and Skoog, 1962)
containing 3 % (w/v) sucrose, 0.8 % (w/v) Type E Agar
suppplemented with TDZ (0.5 μM) adjusted to pH 5.7
(Lata et al., 2008, accepted) The subculturing was done
every four weeks on sterile medium dispensed (25 ml)
in glass culture vessels (4 cm diameter x 9.5 cm high,
baby food jars with magenta B caps).
Encapsulation Procedure
Encapsulation Matrix : Sodium alginate was added
in the range of 2-6 % (w/v) to full strength Murashige
and Skoog's medium (MS) with or without 3 % sucrose.
The solutions were supplemented with 0.5 μM
Thidiazuron (TDZ) and 2.5 μM indole-3-butyric acid
(IBA). A broad spectrum fungicide, Plant preservative
mixture (PPM) in a range of 0.3-0.5 % was added to the
gel matrix for in vivo experiments. For complexation,
different concentrations (25-100 mM) of complexing
agent (CaCl2.2H2O) were prepared in liquid MS medium
containing the same adjuvents as the sodium alginate
matrix but excluding the PPM. Both the solutions were
autoclaved separately for 15 min at a pressure of 1.1 kg
cm-2 and temperature of 121 ºC after adjusting the pH
to 5.8.
Formation of beads : The beads were formed by
dropping explants mixed with sodium alginate solution
into CaCl2.2H2O in a flask, placed on an orbital shaker
at 80 rpm. The resulting beads (0.5-0.8 cm in diameter)
containing the entrapped nodal segments were left in
the calcium chloride solution for 30 min for complexation.
These were retrieved using a nylon mesh and the traces
of calcium chloride was removed by washing with
sterilized distilled water. The encapsulated nodal explants
now called as synthetic seed / beads were inoculated
onto the different planting substrata for growth and
development studies as detailed in results (Fig. 1A).
https://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC3550375&blobtype=pdf

Way ahead of myself of course, but maybe.. eventually. I've already got ideas for encapsulation. lol

 

[Big Sur]

Wow! Way cool. A complete recipe for synthseeds. And a recipe for where to take TC in an MJ plant: the nodes (of course).

For TC in cymbidium orchids we took sections of the pseudobulbs, which could also be divided and planted themselves to propagate that type of orchid. In bamboos I would take TC from nodes in the culms or rhizomes, depending on the genus. Bamboos can be difficult to culture though. For my nursery, I usually propagate my bamboos by planting rhizome cuttings, but I also make whole plant divisions. I have never made synthetic seeds, but it seems rather plausible.

 

[electro gypsy]

I have never made synthetic seeds, but it seems rather plausible.

It sure does. I think we can do it if we can get the foundation laid for TC. I've also been thinking about liquid cultures and something in between. Imagine how easy it would be to simply inject a grow medium with a syringe! It made a major break through in mycology. Changed the whole game. If plant cells can be made as friendly.. who knows