6 Tips to Get In-House Tissue Culture Right

6 Tips to Get In-House Tissue Culture Right

Columns - Tomorrow in Cannabis

Tissue culture can help prevent disease outbreaks in cannabis grows. What to know before setting up a lab in your facility.

May 10, 2021

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Symbiotic rotation, the practice of having all stages of plants ready exactly when you need them, is considered by many to be a safe and predictable practice in commercial cannabis production. This has been, and for the most part still is, how cannabis is produced commercially. Very few commercial cannabis producers start from seed each growth cycle, with the exceptions being those who use auto-flowering seeds/plants and those utilizing feminized seed. (Most hemp fields are grown from seed, not clones.)

The mother/clone symbiotic rotation system can be straightforward—a grower starts with a chosen clone cultivar or selects a preferred specimen from plants grown from seed. Then, the grower designates a given specimen as a mother plant or the chosen female from seed. Then, the goal is to have the clones taken from that mother plant rooted, hardened off, and ready to be moved to a vegetation room at the exact time the current plants inhabiting that room are ready to be moved to the flowering environment, which is at the same time the plants inhabiting the flowering room are ready to be harvested.

All of the above is easier said than done, especially if working with multiple cultivars. It takes a lot of forethought and proper planning to have all stages of growth ready exactly when you need them, not before or after.

Concerns with Common Practices

What many growers did not know (but are becoming more aware of) was these practices leave open the potential for viral infections, insect pests and other diseases. For many years, I’ve heard and read growers blaming detrimental changes in a given line of clone-only cultivars as “genetic drift.” The more likely culprit was and remains Hop Latent Viroid (HpLVd), a virus capable of being passed down through clones and presents with stunted growth, yellowing of the outer edge of leaves (especially in new growth) and reduced yields, potency and terpene content. Recently, test kits for HpLVd have become available to the general public, and the hope is more test kits for different viruses will become available soon.

RELATED: The Threat of Viral Cannabis Diseases

This does not mean all viral infections are HpLVd—as previously stated, there are many viroid/viruses capable of infecting cannabis, but there is limited research on this subject, so the actual number of viral threats is uncertain.

A Different Approach

Growers, especially large commercial cultivators who are concerned about the risk of HpLVd and other diseases, are increasingly considering tissue culture.

At a very high level, tissue culture, or meristem tissue culture, is the practice of excising the newest vegetative material in the form of shoot tips. Those samples then get cleaned/disinfected before being placed into a sterile container with a sterilized replication medium. Many commercially grown plants start from tissue culture, including pineapples, blueberries and tomatoes.

There are multiple benefits to tissue culture:

  1. Growers can start with pest- and disease-free plants every growth cycle;
  2. Growers can eliminate the need for mother plants and a mother plant environment;
  3. Growers can maintain many cultures/cultivars in a relatively small space;
  4. Tissue cultured plants require less space to propagate (compared to the space required for thousands of clones).

Tissue culture also can allow a seed producer with infected seedstock to disinfect the given cultivar or seedstock by taking tissue cultures of both male and female plants and breeding them after the tissue culture process is complete. The resulting seeds from the tissue cultured male and female plants would be viroid/virus free.

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Tips to Get Tissue Culture Right

Tissue culture operations can be done in-house or in partnership with a third-party tissue culture lab. There are many such labs that cater to the cannabis industry. If shopping for a tissue culture partner, growers should ensure that the process employed by the third-party lab is produced in sterile conditions. (Some companies advertise doing meristem culture, but viruses/viroids still can be transmitted if the cells aren’t contaminant-free before being placed in the replication medium.)

Some cannabis producers might want to bring tissue culture in-house. Performing tissue culture on cannabis is a challenge for even the most experienced tissue culture companies. The failure rate can be extremely high, and it can take more than a year to simply get a cultivar in tissue culture, let alone remove any pathogens from it. To do tissue culture at scale well, growers will need large investments as well as someone with extensive tissue culture experience on staff.

For growers considering building a tissue culture lab (whether in their current facilities or as an add-on module), consider these suggestions and necessary materials:

1. A tightly controlled environment is needed to successfully perform tissue culture, especially at a large scale. Mother/cloning rooms might need upgrades to their HVAC and IPM systems, as a sterile environment is required to avoid introducing pathogens to the new plants. Growers also can purchase and retrofit storage containers to act as tissue culture labs.

2. Ensure the interior has the ability to be sterilized (e.g., fiber-reinforced plastic (FRP)).

3. Use tables (ideally stainless steel) with multiple worksites. Each tissue culture lab employee should have their own workstation to avoid cross-contamination.

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4. Each worksite should have a workplace and a laminar flow hood with separate filtration (also called a cell culture hood). A laminar airflow hood has sterilized and filtered air moving from one side of the workstation to the other, making it difficult for contaminants to fall into the sterilized tissue culture containers.

5. Invest in equipment to be sure the growing environment is as clean and safe for tissue culture as possible, including:

  • HEPA filtration on air intake to sanitize incoming air;
  • UV sterilizer on air intake to sterilize intake air;
  • UV sterilizer on interior, design accordingly; (UV sterilizers are used in operating rooms in between surgeries to decontaminate—the same technology can be used in tissue culture labs to save on cleaning labor costs.)
  • Incubator (humid CO2 incubator recommended);
  • Air conditioning of some type that does not contaminate the environment;
  • Centrifuge, which is used to ensure that the tissue culture and rooting media properly settles into the bottom of the container.

6. Consider budget needed for necessary equipment and materials, which also includes:

  • Refrigerator (size is dependent on the scale of tissue culture lab);
  • Freezer (minimum cold temp required: -20° C/-4° F);
  • Microscope or magnification device
  • Autoclave or sterilizer;
  • Water source (both hot and cold);
  • Water bath;
  • Sink;
  • Storage areas (one for extra materials, which can be situated outside of the lab/module, and another for the tissue culture clones, which can be separate from the lab/module but should only house the cultured material).

Once the lab is set up, operating under a symbiotic rotation requires growers replace mother and propagation rooms with cuttings from their tissue culture lab. A plant growth tip is brought into the lab. Then, samples are cut in lengths to allow them to be sterilized. Next, specimens are segmented in a laminar flow hood and placed in a container filled 1/5-full of tissue culture media. (The size of the container varies—one plant can be housed in a single test tube, or five plants or more can be in a single larger container). The container is sealed and placed in an incubator. A separate pod can be designated to be a large incubator. This can be done in an 8-foot by 30-foot shipping container outfitted with minimal environmental controls (e.g., heating, cooling, lighting, etc.)

The process of cloning from mother plants is simple and will continue to be the preferred method of plant replication for home grows as well as smaller commercial operations. But tissue culture rapidly will become the preferred plant replication method of large-scale commercial cannabis producers, because with tissue culture comes predictable, safe, clean and uniform replication of millions of plants. With potentially millions of dollars invested, the alternative for those large-scale growers is too daunting.

Kenneth Morrow is an author, consultant and owner of Trichome Technologies. Facebook: TrichomeTechnologies Instagram: Trichome Technologies k.trichometechnologies@gmail.com