A Primer On Tissue Culture

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Answers to pressing questions on micro-propagation.

April 4, 2018

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Commercial agriculture has many commonalities with commercial and artisanal cannabis, including mass propagation of plants with known genetic qualities for retail production. Whether growing vegetables, trees, herbs or medicinal plants, cultivators share a ubiquitous desire to acquire vigorous plants of known genetics, free of pests and disease.

Cannabis cultivation relies on two things: influx of new genetics with known qualities, and the output or product derived from those genetics. This poses the inherent problem associated with mass cultivation of any crop: Any time growers bring in new seeds, clones or transplants, they run the risk of introducing a new fungus, pest, virus or undesired genetics into their gardens. Even with the most dedicated integrated pest management (IPM) program, pests and diseases can be transmitted unknowingly using traditional propagation methods.

So how do professionals trade genetics, incorporate new ones and protect their existing crops? Five years ago I would have said, “Quarantine rooms and multiple plant redundancies for each lineage.” Now, I will tell you, “It’s all about micro-propagation.”

Micro-propagation, or tissue culture (TC), is the process of cloning the “mother plant” into potentially thousands of exact copies using only a few cells from the stem. That’s right, we can clone from a small mother plant and produce tons of genetically identical, pest- and disease-free clones by using TC.

In fact, micro-propagation has been used for decades in horticulture, from the production of ornamental plantain lilies to banana plants and even giant redwoods.

How Does TC work?

TC is a multistep process. To begin, a section of healthy plant tissue is excised from the plant before washing and sterilization. The plant tissue is then lodged in a gel-like substance with essential nutrients, energy sources and special hormones, and then stored in a test tube. The tissues are kept cool and are progressed through stages of growth using differing hormones, eventually moving into a new gel until the sampling is large enough to multiply.

Once the plant is large enough, it is diced, or “multiplied,” into hundreds of plants. Each of the new cuttings, or clones, undergoes the same washing and gel process until they eventually are given a different hormone to encourage root growth. They are then developed enough to be slowly introduced to the growing environment, or “hardened-off,” and can be slated to be placed into production. These new production plants have identical genetics, are free of pests and diseases and can be produced by the thousands with incredible accuracy.

Technology and the internet have provided the proper resources to allow cannabis cultivators with no micro-propagation experience to get started. It only takes a couple hundred dollars to purchase a start-up kit and perform several tissue culture clones. The kits contain a small number of vessels and enough hormones to run a few clones before needing more equipment and supplies.

The kit method needs to be modified and improved upon for a small-scale operation that is cloning between 200 to 500 plants weekly—anything larger than that would need to be farmed out to a micro-propagation company to be the most economically efficient. The minimum scale at which it makes economic sense to use an outside group to handle your tissue culture up to the hardening-off stage is probably around 1,000 clones weekly.

Fair warning to those trying the in-house method: You need to have a strong grasp on a clean procedure, as well as keep some simple household items, to minimize and prevent contamination. All in all, in a couple of hours, a TC newcomer can set up a clean area and begin successfully multiplying via tissue culture.

What Issues Might Growers Expect?

A problem with cloning is “sporting”—genetic mutations caused by hormonal influence—and that can become detrimental. Sporting occurs when mother plants undergo certain stressors that allow for a systematic divergence from the original genetic code. Because this originates in the mother plant or when cutting of the clone, both TC and traditional cloning are susceptible to sporting.

To address this, it is important to obtain a baseline genetic blueprint from a company that does genetic mapping, such as Phylos Bioscience, so you may periodically check for mutations, or what most people incorrectly call “genetic drift,” and select an alternate, healthier branch for multiplication.

There is a difference between mutations such as sporting and genetic drift. Genetic drift is a specific term that implies recombination of genetic material from two parents. Because we are cloning from only the female sex, we are technically not observing drift. Genetic drift drives a population toward a single form over time, while sporting might depend on how the mutation affects the alleles (the copies of genes that govern the genetic code). If the new plant is used for asexual cloning (standard or TC) or sexual fertilization (seed production), you could lose genes of interest. Either way, there are inherent risks to propagating your crop and potentially losing your genetics, so always maintain redundant crops for those strains you cannot afford to lose.

The main drawback to TC is cost. Setting up a large-scale production facility would reach the multimillion-dollar mark today. Industry and technology will need to continue to improve and reduce costs so that one day TC is affordable for all.

Another drawback to TC is transporting requested strains from the TC facility to the growers in a timely fashion. These transportation issues become incredibly challenging to maintaining crop schedules given cannabis crops can take more than two months to reach hardening stages, then spend four weeks in vegetative growth, and seven or eight weeks in flower.

Nastasic | iStockPhoto

Why Use TC?

Traditional clones are asexual copies of the mother plant from which they came. They tend to be 3 to 6 inches tall with some foliage atop a stripped-down stem, somewhat resembling a small palm tree. The clones sustain from the stores of nutrition in the stem and foliage as well as the humid environment and the substrate they occupy. This, along with an imbalance in hormones, signals the cutting to create roots, and over the next few days they are ready for potting in larger pots.

The beauty of traditional cloning is you get a living plant in less than two weeks. On average, one of our sites in the high country of Arizona has transplantable roots on our healthy clones by day seven, although the unhealthy clones can take more than three times as long, especially if re-vegging a flowered branch. (Re-vegging can happen when the genetic lineage is accidentally flowered before a new generation is started.) However, the problem with cutting clones is that their quality depends on the condition of the mother plants. If the mom has mites, a virus, mold or nutrient deficiencies, the clone will have to combat this while staying alive and making new roots.

TC, on the other hand, has a built-in process that eliminates pests and diseases while preparing the plant tissue for cloning. While the entire process can take months and needs a high level of logistical organization to know and track the varieties needed for cultivation, the redeeming quality worth its weight in gold is that TC sterilizes the next generations. By using TC, a grower can be certain the generation to come will start free of pests and diseases. Considering some of the most devastating pests in the industry can hide behind the width of a human hair and are smaller than dust mites, you want to be certain you are not propagating them around the site, or worse, trading them.

Aside from the above position on TC and traditional cloning, the absolute supreme reason for paying attention to TC is the ability to maintain a genetic lineage in a suspended animation for prolonged periods of time (also known as “tissue banking”) without taking up large amounts of space to keep it protected from pests and diseases.

Tissue banking is the best way to preserve genetics for decades without having to dedicate production space and without having future knowledge about its chemotype. Cannabis is in its infancy from a science standpoint, and we will no doubt learn more about the minor cannabinoids and terpenes that govern the experience consumers receive as well as the medicinal value patients obtain from the plant. TC provides an incredible system for banking genetics, redundancy and potential future patents, and it encourages legal trading of cannabis cultivars in a strictly regulated market. When we learn more, there may be varieties no longer in production that bear chemistries we are now interested in. Due to the nature of seeds and cannabis, saving seed is not always a sure thing when looking for a specific chemotype. Only a true clone can provide exact genetic duplication.

Eric Culberson, RN is the director of cultivation for Columbia Care, a national brand of medical cannabis cultivation businesses. In that role, he conducts cannabis development studies with an emphasis in sustainability.