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How to Tissue Culture Cannabis

By Aimee O'Driscoll

Published: Sep 06, 2022   

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How to Tissue Culture Cannabis

Many cannabis producers are looking for ways to improve their yields and increase the quality of their product. One way to do this is through tissue culture. Tissue culture is a process by which living cells are grown in a controlled environment. Cannabis tissue culture involves micropropagation, where producers grow plants from tiny samples of viable tissue.

In this article, we explain more about tissue culture, the benefits of using it to grow cannabis, and key factors to consider before creating a cannabis tissue plant culture. If you’re interested in trying tissue culture yourself, we provide a step-by-step guide on how to get started.

What is tissue culture and what are the benefits?

Tissue culture is a plant propagation method involving tissues or cells being removed from a live or freshly harvested plant and grown in a sterile environment. This technique produces clones (genetically identical copies) of the original parent plant. It can be especially useful for strains that are difficult to propagate from seed or for growers who want to replicate a plant with desirable properties. Tissue culture can help preserve valuable genetics and can be used to create uniform plants that are less susceptible to disease.

Tissue culture allows cannabis growers to create new plants from existing ones without going through the process of nurturing cuttings. When producing clones from cuttings, the plants must be kept in a controlled, disease-free environment until they sprout roots. This can take up lots of space and increase production costs. Tissue culture yields similar results to using plant cuttings but takes up far less space and can lower expenses.

Key considerations in creating a cannabis plant tissue culture

Tissue culture involves growing samples in a micropropagation media that includes the exact nutrients that the plant needs. The ideal composition of the media is one of the major considerations in cannabis tissue culture and will depend on features of the plant starting material.

“When approaching tissue culture, the first consideration we make as scientists is the age and condition of the starting material and if it is a healthy or unhealthy donor plant,” David Critzer, cannabis tissue culture expert and instructor of Plant Cell Technology’s Tissue Culture Master Class: Cannabis, tells Analytical Cannabis.

“If the starting material is generally healthy and free from internal pathogens and we want to make genetic copies of the source plant, or even if we wanted to remediate surface pathogens like molds or bugs, we would use micropropagation to make additional plantlets.”

For unhealthy material, things are a little more complex, and an additional step is needed to produce non-contaminated tissue.

“If the source plant had internal pathogens such as viruses, viroids, or systemic fungi that are unable to be remediated with basic surface sterilization, we would consider amending antivirals, antimycotics, or antibiotics into the micropropagation media as a pretreatment to grow selected plantlets on,” says Critzer. He explains that the next step is a meristem isolation, where a microscope is used to isolate non-vascularized tissue that is free from contamination. This is then allowed to grow back into a full plant in a sterile environment.

You also need to consider the time required to create cannabis plant tissue culture. “Micropropagation (node culture) can be as fast as five-to-six weeks to rooted/acclimated plants after initiation,” says Critzer. “Meristem regenerations can range anywhere from 14 weeks up to a year to regenerate into a whole plant, depending on the size of the starting material.”


Verónica Codesido, PhD, co-founder and cultivation accountable for MIFCO Biosciences, explained meristem culture in more depth during her presentation at the Analytical Cannabis Expo Europe Online 2021.

Equipment required for creating cannabis plant tissue culture

Once you’ve decided on your method, you’ll need to ensure you have all the required materials available. Media culture ingredients are of particular importance. These can vary depending on the starting material, but the Murashige & Skoog (MS) Basal Salts Mixture forms the basis of most plant tissue culture growth media. This is mixed with sugar and distilled water, and the pH should be adjusted to 5.8.

While the liquid may be used as a growth media, adding agar will thicken the solution to a gel material ideal for use in petri dishes or other shallow vessels. You may also choose to add other ingredients such as plant hormones. For example, certain cytokinins can promote shoot formation, while select auxins can encourage rooting.

Common pieces of equipment used in cannabis tissue culture include a laminar flow hood, an autoclave, a stereo microscope, an orbital shaker, a milligram scale, a magnetic stirrer, and a pH meter.

Step-by-step micropropagation of cannabis tissue

As Critzer explains, the four main stages of micropropagation include:

  • Stage 1: Surface sterilization and aseptic initiation
  • Stage 2: Shoot proliferation and multiplication
  • Stage 3: Rooting or preplant stage
  • Stage 4: Acclimatization and hardening

Let’s look at each of these in a bit more detail:

Stage 1: Surface sterilization and aseptic initiation

The first step in tissue culture is surface sterilization to prevent contaminants from infecting the plant tissue. You should also ensure any tools are sterilized, for example, using isopropyl alcohol or an autoclave.

Next, you can prepare your tissue culture media for the shoot multiplication and rooting stages. The media should be sterilized using an autoclave at around 121°C (249.8 Fahrenheit) and then cooled. Once prepared, it is poured into sterile containers; tubes or petri dishes are commonly used.

Now it’s time to collect your plant material. Samples can be taken from various parts of the plant, including buds and shoot tips. Depending on the sample type and whether or not it came from a sterile plant, it may need to be washed and sterilized. The tissue can then be placed in the shoot multiplication media.

Stage 2: Shoot proliferation and multiplication

Once samples are placed in the media, they need to be maintained under ideal conditions. This typically comprises a temperature of 25°C (77°F), 60% humidity, and fluorescent light. During this stage, shoots proliferate and roots start to form.

Stage 3: Rooting or pre-plant stage

The next stage in tissue culture is the rooting or pre-plant stage, where shoots are transferred into the rooting media and kept under ideal conditions. Once sufficient roots have formed (after about six weeks), the plantlet is ready to be transferred into soil.

Stage 4: Acclimatization and hardening

The final stage in tissue culture is acclimatization and hardening. This is when the plant is slowly introduced to different environmental conditions. For example, the plant may be exposed to different amounts of light or varying temperatures. This process helps the plant adjust to its new environment and ensures it can thrive once it is transplanted into its final growing vessel.

Common challenges in cannabis tissue plant culture

Cannabis plant tissue culture can run very smoothly and you might be surprised at the speed with which you achieve desirable results. But it doesn’t come without its challenges.

“Vitrification, aka hyperhydricity, is an issue exclusive only to tissue culture plants and is the most common problem scientists must overcome when developing protocols,” says Critzer.

Hyperhydricity is a condition in which plants have excessive water in their tissue, leading to wilting, yellowing leaves, and stunted growth. It can often be caused by overwatering, poor drainage, compacted soil, or fungal or bacterial infection.

“After that, we are commonly seeing hops latent viroid, cannabis cryptic virus, lettuce chlorosis virus, and beet curly top virus in the cultivation space depending on where you are in the world,” advises Critzer. He adds that cultivation style factors into your susceptibility to different vectors of transmission.

Conclusion

Cannabis tissue culture offers a relatively fast and efficient method to produce large numbers of plant clones, helping you preserve valuable genetics. Certain techniques can also help you produce disease-free versions of contaminated plants.

While there are several potential challenges with cannabis tissue culture, if you take the time to ensure you provide the plant tissue with a sterile environment, the right nutrients, and proper care, you should be able to successfully produce healthy clones.


Aimee O'Driscoll

Freelance Science Writer

Aimee is a freelance science writer with over a decade of experience as a development chemist. She has written for Analytical Cannabis since 2020.

 

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