Cannabis Science

22 April 2021

What is Cannabis Tissue Culture?

Plant tissue culture, also known as micropropagation or in-vitro propagation, is a method of creating plant clones using living tissue such as a piece of a stem, leaf, or flower. The plant tissue is placed in an extremely sterile environment/medium, which allows it to multiply its cells and grow into a new plant. 

Sterile conditions are of utmost importance in the micropropagation process, as the tissue cuttings (known as “explants”) are extremely fragile and may not survive an unclean culture medium.

Cannabis tissue culture shares many similarities with cloning techniques: cuttings taken from plants are induced to root, which creates a plant clone that is genetically identical to its mother plant. The most important factors that separate tissue culture from traditional cloning methods are the size and number of cuttings, the level of sterilisation involved in the process, and the sophistication of equipment needed to keep cuttings sterile and healthy, both of which raise the barrier to access significantly for cultivators.

Advantages of tissue culture over traditional cloning methods

Micropropagation has made the large-scale production of viable individuals of many plant species possible and plays a key role in the cannabis industry and modern agriculture, horticulture, and the food supply. It supports crop production by ensuring a stable supply of production-ready phenotypes, streamlining, and facilitating breeding of hybrid varieties, and eradicating pathogens from plant varieties. A growing number of legal cannabis cultivators in California use tissue culture to preserve their proprietary genetics.

Compared to traditional cloning, cannabis micropropagation can deliver a larger amount of identical plants. Over several weeks, dozens, hundreds, or even thousands of plantlets can be produced from a small amount of plant tissue. It would be difficult to achieve the same level of production using traditional cloning methods as mother plants need time to recover after cuttings have been taken.

Cannabis growers may find the speed of production of tissue cultured cannabis plants desirable for reducing the amount of time between generations for any breeding projects, meaning any new commercially viable strains can be brought to market quicker and with more regularity. Similarly, tissue culture propagation also produces clones that are free of viruses and diseases, which may transfer from mother to clones in traditional methods of cloning.

The stabilisation of cultivars is a considerable advantage of this technique as it will provide growers with security and peace of mind when it comes to planning their next growing project.

There has been substantial evidence on the successful effect of micropropagation “rejuvenating” genetics. When plants are propagated from tired mother plants or strains that have been replicated via traditional cloning for a long time, the resulting plants have grown with more vigour and resistance to pests and disease as well as with larger yields, more concentrated cannabinoids THC and CBD (as well as others) and stronger terpenes.

This is not a guaranteed result, however, and even tissue cultures completed in the most sanitary environments using the best tools have failed if the plant that is inducted into the process is too old, diseased, or otherwise unfit.

In addition to crop-planning advantages, growers will be pleased to find savings on the space required to successfully run a culturing operation. Overall, tissue culture cuttings and plantlets occupy a much smaller footprint than traditional clones. Tissue culture propagation of plants can also be carried out at any time of the year, meaning growers are not tied to a specific season or weather for taking clones of their plants.

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Tissue culture is sterile cloning

The most important factor in determining the success of tissue culture is complete sterilisation in all stages of the process. What this means for the grower is properly sterilising tools, vials, and cutting equipment, as well as the living plant tissue to be cultured once it has been cut. A clean growing environment is ideal not only for plantlets to grow in, but it’s also the optimal situation for any undesired bacteria, micro-organisms, or fungi, any of which would derail the tissue culture process and kill the extremely sensitive and fragile plantlets.

Tools for tissue culture are sterilised using heat or pressure, while cuttings (also referred to as “explants”) can be disinfected using at least 70% alcohol and a 1-3% bleach solution. Sterilisation is the most time-consuming (and expensive) stage of the process and is the trickiest stage for growers to dial in, depending on their equipment and experience.

Tissue culture is very involved and requires different tools and containers for each stage of the process. It is helpful for the grower weighing the pros and cons of a tissue culture workflow to consider that at each stage, sterilisation will be necessary, or the process may not be successful. Something that seems as harmless as breathing too close to the explants or medium may introduce bacteria that would hinder or completely ruin the process.

A workflow for this process looks something like this:

1 . Select the plant to make cuttings from

2 . In a sterile environment and using sterilised tools, create explants by cutting tissue samples from the original plant.

3 . Trim away superfluous plant material from explants, leaving smaller stems with a single node.

4 . Sterilise explants in separate solutions of alcohol and bleach to destroy any bacteria found on the explants before introduction to the growing medium. This is the most effective way of preventing contamination.

5 . Place explants inside sterilised containers with the correct growing medium. This will typically consist of a formulation Murashige & Skoog medium (also known as MS solution), which the grower adjusts the pH of to 5.8 and can be made at home using recipes or ordered online as a premade powder. MS solution contains inorganic compounds such as mineral salts needed for plant growth and development, organic nutrients such as vitamins and amino acids, growth hormones that help promote stem growth and root development, and gelling agents such as agar derived from red seaweed to help establish the cultures.

6 . Let explants grow into plantlets in their mediums by ensuring optimal conditions and providing additional nutrients if required.

7 . Explants should be kept in conditions of 24-29C and on a photoperiod of 16/8. Ensuring adequate airflow will prevent overheating from grow lights.

8 . Plant growth regulators can be added to the growing medium to stimulate tissue growth.

9 . Introduce rooting hormones to induce plantlets to grow roots.

10 . Transfer plantlets to their growing medium for the next stage of development.

The timeline for micropropagation of cannabis plants using tissue culture is generally 12-14 weeks from explant to production-ready clones ready to plant. The grower considering this process should keep in mind that failure is still possible even if their workflows and areas are bulletproof and the highest levels of sanitation are achieved.

Cannabis cutting in test tubes known as cannabis tissue culture method

Sterile environments and plant growth regulators

Cannabis cultivators are generally resourceful and attentive growers with limited laboratory skills but who are willing to experiment may find other ways to close the gap to deliver successful tissue culture propagation.

Continuing with the general theme of sterilisation, a clean environment can be made at home using a suitably sized terrarium container turned on its side. After sanitising the inside using bleach and alcohol, the grower can seal the opening with plastic. When the grower is ready, they can cut holes in the plastic to put their (gloved) hands through.

Explant growth can be manipulated by introducing hormones to promote and regulate the growth of different tissues. The first development milestone an explant will achieve after successful induction into a sterile medium is the formation of the callus. This is a mass of unorganised plant cells that establish before continuing to become shoot or root tissue.

Auxins and gibberellins promote cell division and elongation, which leads to the development of plant callus. Cytokinins can be introduced into the medium to promote stem and shoot development or to hinder root development if necessary. Abscisic acid can promote or inhibit callus growth.

While plant growth regulators may seem intimidating to consider, they often come in combination solutions that can be easily purchased online.

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Post author
Martin
Martin is a production horticulturist with experience in commercial cannabis cultivation and sustainable farming from his time with Emerald Cup Award-winning farmers Esensia Gardens in northern California's Emerald Triangle.
See more from Martin

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