Synergy Genetics - Tissue Culture Q & A

Advanced Techniques
#1

Hi Folks, SCJedi here to offer up a space where we can chat about the ins and outs of cannabis tissue culture. This is a safe space and there are no dumb questions.

I teach tissue culture classes in the Sacramento, CA area but would like to provide some basic information here on the primary types, components, and techniques that are required for success as well as a platform to ask questions for those heading into the tissue culture rabbit hole.

This will be BASIC and INTRODUCTORY. If you have desires about advanced techniques I will point you in a direction that you can research on your own. Somewhere between Intro and Advanced, I expect that you can download, read, and comprehend scientific papers so make sure to add that tool to your toolbox in between the two. I can also cover it as a topic here.

First things first, your success is determined by your personal tolerance for contamination. If you can get 1 out of 10 tries correct and that is fine, then you have succeeded. If you want 99 out of 100 to be uncontaminated and you hit that mark, then you have succeeded. Only you know what success looks like. With that said, consumables cost money, and your time costs money, so do your own cost-benefit analysis, please.

Tissue culture comes in two primary flavors; nodal and meristem. While there are some similarities these two techniques have advantages and disadvantages

Nodal: (Primarily used to store and rotate clean mother stock)

Advantages:

  • Fast and efficient multiplication at scale
  • Sterile and disease-free (if mother stock is proven pathogen-free)
  • Genetic and phenotypic uniformity
  • Reduced space requirements vs. traditional mother stock
  • Reduced risk of maintenance of high-value genotypes

Disadvantages:

  • Time (Growth condition optimization process R&D)
  • Costs (Specialized Lab Equipment like a hood, scope, and autoclave)
  • Skilled Labor Learning Curve (Learning/practicing aseptic technique)
  • Losses (Contamination)

Meristem: (Primarily used to clean mother stock)

Advantages:

  • The same benefits of nodal
  • Have an isolated vascular system so lower risk of pathogens

Disadvantages:

  • Much longer to culture ~10-12+ months start to finish
  • Can still be susceptible to pathogenic infections - Mitovirus
  • Requires greater technical & precision skills
  • Mutation (somaclonal variation, i.e. not true to type)

There are multiple “stages” to plant tissue culture (PTC) that are in a Roman numerical order but DO NOT need to occur in this exact order, or need to occur at all.

Stage 0: Cultivation, Selection & Preparation of Explants
Stage I: Establishment of Explants
Stage II: Shoot Multiplication
Stage III: Rooting
Stage IV: Acclimatization

With that said, let’s dive into some of the basics.

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#2

Media

Media comes in a multitude of forms and is your primary tool to tell your plant’s parts (explants) what you want them to do and to provide the perfectly optimized environment to do it in.
Media varies in viscosity depending on what your experiment dictates. It can be liquid, soft, semi-solid, solid, etc.

Media consists primarily of a few basic ingredients that do not vary much:

  • Gelling agent (agar agar, gellan gum, gelatin, etc.)
  • Basal salts (nutrients)
  • Carbon (sucrose)
  • Plant Growth Regulator (PGR, plant hormones)

That is it.

Basal Salts like MS/DKW/WPM, etc are nutrient basal salt mixtures that are equivalent to the hydroponic salt mixtures that growers have been developing over time.
Plant tissue culture (PTC) scientists have done the same thing for decades and decades. These are primarily developed, or optimized, to tissue culture for a particular type of plant or tree.

Gelling agents vary in concentration to determine how “solid” you want the matrix of your media to be. The higher the concentration of the gelling agent the ‘stiffer’ the media.

Carbon is a media requirement because explants DO NOT photosynthesize the way a whole plant does. Your explant pulls carbon from the media instead of from CO2 in the air. Plants need carbon to survive and flourish.

Plant growth regulators, or PGRs. This is a load road but I will make it short for this thread. These may also be referred to as phytohormones but we like PGR since we are acronym nerds. Plants create PGRs naturally and they are endogenous. Scientists have synthesized many to make them cheap and more readily available.
The two primary classes of PGRs used in PTC are cytokinins which cause plants to grow shoots and auxins which cause plants to root. Shoot (Stage II) and Root (Stage III), those are our two primary goals, in vitro.

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#3

I am going to add a few placeholders to add some kind of things later

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#4

I am adding a few common introductory scientific papers. these will talk about some of the more “novel” approaches to media and PGRs, more specifically, TDZ and m-Topolin. The big takeaway from reading these is the look at what they are using, how they make their media, and what worked for them.

A Micropropagation System For Cloning of Hemp (Cannabis Sativa L.) by Shoot Tip Culture - Wang.pdf (118.2 KB)

Thidiazuron-induced high-frequency direct shoot organogenesis of Cannabis sativa - Lata.pdf (276.9 KB)

In vitro mass propagation of Cannabis sativa L. A protocol refinement using novel aromatic cytokinin meta-topolin - Lata.pdf (2.7 MB)

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#5

The science is above my pay grade but I’ve been really interested in the process for years. Thanks so much for sharing your experience with everyone.
My personal scientific method is akin to "Screw around and find out " lol

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#6

Happy to tag along, Subject of the future, in my opinion. I have the Media, Pressure Cooker, Tubes, making my own Hood, installing appropriate-size Filtration. Looking to begin in Late Spring. Have the Topic on “Watch”. THANKS for your insight, SS/BW…mister :honeybee: :100: :pray: :smiling_face_with_three_hearts:

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#7

Screw around and find out = Research & Development. Also, nothing is above anyone’s pay grade. I started by doing this at my kitchen table with an upside down tote. You can too so don’t overthink it. This is plant science, not rocket science.

Coolio, you are on your way so let’s make this thread your guide!

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#8

Thank you for putting this together, I have always wondered what tissue culture is all about. Fascinating science.

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#9

I’ll be lurking in the back taking notes… :+1:

Cheers
G

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#10

Many thanks SC_Jedi for the post and info!!!

I really would like to take this opportunity to ask you few questions and maybe enter into the topic.

  • Benzlaminopurine (BAP) I Bought it…is it needed?
  • PPM I didn’t buy as it is very expensive ( BAP as well). I read some protocols are not using PPM.
  • Protocols: any link to free sources? Protocols specific for different type of explants. leaves is more difficult that stems? Could be root?
  • Tricky one: what’s your opinion about mushroom incubator ( which simulate a laminar flow but without the HEPA filter, etc,etc,etc,etc) I like Plants in Jars YTVideos. + Kitchen well clean + pressur cooker as autoclave or microwave as steriliser.
    Which % of success would you believe I could achieve?
  • Timing. After success can you control the grow to reduce or accelerate the passage to acclimation? Any link about timing from a leaf to be a 5’ cut. How long does it take? Can you use tissue culture and fridge to store genetics for long time ( 1-2 years before need to change medium)?
  • DIY/Homemade: any link/info appreciated.

Many thanks!!!

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#11

Been journeying down this rabbit hole myself recently.l

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#12

Thank you for starting this thread, I’m going to do some reading!

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#13

Here you go, I posted in-line, in BOLD:

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#14

Thank you so much for this topic ! One quick question. Can an agar-malt solution ( designed for mushroom culture) be used as a base medium for plant tissue culture? Thank you :grin:

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#15

You would not want to use MEA in plant tissue culture. Plants feed off the carbon from sucrose. Fungus feed off MEA and use malt as a sugar source.

One thing to consider though if you have mycology experience is that you already have a jump start since you know sterile technique!

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#16

No words SC_Jedi, very much appreciated all your answers and tips.!!Very clear and I will definitely follow them in some experiment.

Note: sorry 5 inch not foot

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#17

Dear @SCJedi,

If possible I would like to back to you with two questions:

  • Lighting: what’s the minimum light requirements you consider is needed for the tissue culture ? Light cycle as well would be appreciated.
  • Temperature and environmental conditions.
    Again minimum requirement with the intent to preserve genetics more than clean mother and clone back ( if this makes sense).

Many thanks in advance

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#18

Hi @Rhizome,

Light at 50umol and 18 hours of light are fine since these are all veg cloning lights work great. Most papers say 25 degrees C which is about 77 F. I aim for 75-80.

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#19

Many thanks!

I will try in few weeks or so…I hope to post some pic after for your comments and check results.

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#20

I’m at the beginning of taking a deep dive into TC and this is great. Thank you so much @SCJedi for the information. Watching intently! :eyes::grin::pray:

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