Cannabis Plant Tissue Culture: What is it?

How is tissue culture different from cloning and what is it.
10 December 2020
14 min read
Cannabis Plant Tissue Culture: What is it?

Contents:

Tissue culture consists of taking living tissue from a plant and growing that tissue into a full plant in sterile conditions. This process dates back to 1885 and it’s relatively new in the cannabis but is highly effective and can be used with most plant species.

1. The History Of Tissue Culture

The history of tissue culture dates back to 1885 when a German zoologist attempted to cultivate tissue from a chick embryo but wasn’t until 1907 when an American zoologist successfully cultivated a frog’s nerve cell.

 

Cannabis plant tissue culture: history

Tissue culture was first attempted by a german zoologist who tried to cultivate tissue from a chick embryo.
 

Later on, in 1910 a French surgeon improved this technique and defined what tissue culture was, after the technique was defined and established, several other experiments succeeded and enabled other researchers to grow stem cells under artificial conditions which led to more knowledge about human biology and great progress in regenerative medicine.

But, you might be asking yourself How does this relate to cannabis?

2. What is Plant Tissue Culture And How Does it Work? 

Tissue culture is exactly what was explained above, so basically, getting just a few pieces of plant tissue or cells and developing hundreds or even thousands of identical clones from it, this method allows growers to preserve a certain strain almost forever while needing minimal space and effort

As regular cloning, tissue culture starts by having a small part of the plant you want to clone, unlike regular cloning, this tissue sample is trimmed, sterilized, and once it’s cleaned, goes into a nutrient culture that not only contains nutrients but also hormones and sugars.

 

Cannabis plant tissue culture: what is it?

Unlike cloning, tissue culture can be done with just a few plant cells.
 

This nutrient culture depends and is basically crafted for each tissue culture because some nutrients and hormones will trigger certain stages of development so if you want to trigger growth, root development and/or multiplication (when the plant is large enough to do so) you’ll have to provide a specific nutrient culture for the development you want.

So, basically, you would have a bit of tissue and once it’s large enough, it can be cut into hundreds of pieces to be cloned separately, if that’s what you wish to do, once these tissues are large enough to grow, they’ll be fed a nutrient culture that will encourage them to root and then grow.

 

3. Tissue Culture vs Cloning

As the cannabis industry evolves, commercial growers are looking for the best way to get more yields and potency in less time.

When possible, growers are making a change from cloning to tissue culture but why?

Cloning 

As you may already know, cloning consists of taking cuttings from a plant and waiting until it has developed roots to be able to grow it, this clone will become an exact copy of its mother.

 

Cannabis plant tissue culture: cloning vs tissue culture

Cloning vs tissue culture.
 

But clones can be unreliable as seedlings because it can be quite hard to have a good survival rate and not only this, they are susceptible to pests, infections, diseases, and as generations go by, it’s easier and easier to get genetic mutations.

Now with tissue culture, it’s quite different.

 

Tissue culture vs cloning

Tissue cultureCloning
More expensive than seeds and clones.Less expensive than seeds.
Higher survival rate if done properly.Lower survival rate.
Can get hundreds or even thousands of clones from a single leaf.Can get hundreds of clones from a single plant.

 

Plant tissue culture

The process of tissue culture is relatively simple but it requires a lot of control to ensure the right conditions are kept, tissue culture laboratories need:

Sterilization

Tissue cultures require an ideal environment for the plants to grow so this environment should deliver everything a plant needs in a form that’s easy to absorb but that environment is also ideal for bacteria and harmful microorganisms so everything should be sterilized.

This sterilization is vital because fungi or bacteria may end up killing your plants, so not only the lab should be sterilized but also the tools, equipment, and also the plant tissue (aka “explant”).

 

Cannabis plant tissue culture: cloning vs tissue culture

Just like with cutting, tissue culture requires certain conditions that should be followed strictly.
 

Aseptic environment  

It’s vital to have the complete process in a completely sterile environment so, in ideal conditions, a special chamber is created so you can touch the equipment that is already sterile.

This special environment is used every time you need to expose anything such as tools, equipment, or tissue to the air.

Special grow medium

With plant tissue culture, a sterile medium is made to provide the plant with nutrients, sugar, water, and a place to grow, this medium provides everything your plants need to survive until it turns into a whole plant.

This medium can be made at home with a pre-made powder, sugar and distilled water pH’d at 5.8, so although it’s relatively simple, it takes a lot of safety measures.

 

The pros and cons of tissue culture

ProsCons
Plants can be grown in a short amount of time.Requires more labor and money.
Only a small amount of tissue is needed.Less resilient due to the sterile environment.
The new plants are more likely to be disease-free.Tissue needs to be thoroughly checked for abnormalities.

 

5. The 5 Steps Of Tissue Culture

Here are the 5 simple steps of plant tissue culture in a laboratory, just have in mind that this is a resume of the whole process.

  1. Select the plant you want to use;
  2. Place tissue into the sterile environment, remove a piece, clean it and let it grow in the proper medium;
  3. Ensure proper conditions and let the tissue multiply (typically takes 14 days);
  4. Force plant to root;
  5. Transplant to final container and let grow.

Now, if you want a more detailed step-by-step and how to make it at home, here’s a guide, just have in mind that you will need expensive tools so even though it’s DIY, you need special equipment.

 

How to plant tissue culture at home

If you're interested in a guide on how to do plant tissue culture with cannabis, here's a detailed step-by-step, as said above, you will need special equipment but it's totally possible to do it at home.

Initiation phase (1-2 weeks)

Step 1

Turn the flow hood on and run it for at least 30min before starting, if you don’t have a laminar flow hood, use an alcohol burner/lamp and work close to the flame.

 

Cannabis plant tissue culture: step-by-step guide

Make sure you sterilize everything before strating.
 

Step 2

Sterilize the flow hood with 70% ethanol but do not spray it directly to the filter.

Step 3

Turn on your sterilizer and let it warm up for 20min.

Step 4

Sterilize a pack of paper on a dry cycle, then sterilize the vessels, water and tween on a slow cycle.

Step 5

Then to prepare the medium, get a container, fill it with reverse osmosis, distilled water or Deionized water (ideal). If you’re making 1L of medium, add 900ml of water.

After you have the water ready, add Grow nutrients until you reach an EC of 1.5, then add 30g/L of sugar and adjust the pH of the solution to 5.7, then add 6g/L of agar and put in in an autoclavable bottle but do NOT overflow it.

Run the bottle on the sterilizer for 15min and then pour around 50ml in each sterilized vessel you’ll be using.

Step 6

Now that you have the medium ready, prepare a solution with 0.5% bleach and 0.1% sterile tween 20, if you only have 2.75% bleach, add 181ml per 1L.

 

Cannabis plant tissue culture: step-by-step guide

make sure that even the plant material is sterilized before beginning the process.
 

Step 7

Fill a container with a small amount of water, take your cannabis material, and place it in water, make sure the plant material is healthy. If you’re storing the plant material for more than 1hr, place it in a cooler with ice and try to get it in water as soon as possible.

Step 8

Get a sealable sterilized container and fill it about 80% with the bleach/tween solution, then add the plant material and seal it.

Step 9

Shake gently for 20 min and then shake vigorously for 20 seconds after the initial 20min.

Step 10

Transfer the plant material to another vessel filled with 80% sterilized water and shake gently for 5 min and repeat 2 more times.

 

Cannabis plant tissue culture: step-by-step guide

After everything is done you will have to place your explants in a vessel and let them grow for a minimum of 3 weeks.
 

Step 11

Use sterile forceps to transfer the plant material to a sterile piece of paper to absorbs the excess water and then place one piece of plant material per vessel.

The plant should be in an upright position into the vessel and then placed on a rack at 2°C under an 18/6 light cycle for at least 3 weeks.

After your plants are ready, you’ll successfully grow a clone and it’s now ready to be grown in your medium of choice, either outdoors or indoors.

 

Chemical Composition Guideline 

Have in mind that there are different types of media, the minerals and amount may change according to the type of medium you’re using, this guide has been written using the Murashige-Skoog medium as an example so make sure you read and find out which ingredients and the quantity that is preferred in the medium you're using.

 

Macronutrients

Inorganic Nitrogen25000-60000µmol/L
Potassium20000-30000µmol/L
Phosphorus, Magnesium, Sulphur, and Calcium1000-3000µmol/L

 

These are the essential nutrients your cannabis plants need in high amounts and are essential in tissue culture, without them your plants won’t be able to grow green matter.

 

Micronutrients

Cobalt and Copper0.1µmol/LZinc5-3µmol/L
Iron and Molybdenum1µmol/LManganese20-90µmol/L
Iodine5µmol/LBoron25-100µmol/L

 

These are the essential nutrients your cannabis plants need in lower amounts but are also essential in tissue culture, without them your plants won’t be able to grow properly and will surely show deficiency symptoms.

 

Vitamins

Thiamin0.1-10mg/LPyridoxine0.1-10mg/L
Nicotinic Acid0.1-5mg/LMyo-inositol50-500mg/L

 

These vitamins are essential to aid in your plant’s metabolism, especially when talking about tissue culture so make sure you provide them and in the right quantities.

 

Amino Acids

Casein hydrolysate0.25-1g/LAsparagine100mg/L
Glycine2g/LL-Arginine10mg/L
Glutamine8000µmol/LL-Tyrosine10mg/L

 

 

7. In Conclusion

Cannabis tissue culture may seem quite hard but it's relatively simple, you just have to make sure you are maintaining the best conditions and take all the safety precautions to ensure the highest survival rate.

We know that this technique is relatively new and can be quite expensive so not all growers may have access to the equipment needed but if you work or have made your own tissue culture lab at home.

Please share your knowledge with fellow growers, leave a comment in the comment section below!

 

 

This post was most recently updated on February 8, 2021.

 

 

External references

  1. Plant Tissue Cultures. - Kärkönen, Anna & Santanen, Arja & Iwamoto, Kuninori & Fukuda, Hiroo. (2020). 
  2. Nanotechnology and Plant Tissue Culture. - Tariq, Amina & Ilyas, Saiqa & Naz, Shagufta. (2020). 
  3. Plant Tissue Culture Media, Recent Advances in Plant in vitro Culture. - Annarita Leva and Laura M. R. Rinaldi (2012).

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10 December 2020
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