Plant Tissue Culture: Cloning of the Future?

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Cannabis Plant Tissue Culture at Cultivar Syndicate. Photo courtesy of Oleg Zharsky.

Plant tissue culture is a method of propagation that has been sprouting in popularity throughout the cannabis community as an alternative to cloning. But is tissue culture really viable as an alternative to conventional cloning methods? Is it possible to successfully root plant tissue cultures if you’re a home-grower? We’ll answer all these questions and more in this Grow Guide chapter on plant tissue culture.

I’m not going to lie, tissue culture comes off as intimidating to the average cannabis farmer like myself. The image often painted is scientists wearing white coats and goggles, looking at tiny test tube plants in professional laboratories. But tissue culture methods can actually be applied in a variety of environments and situations — from the curious gardener with a modest home-grow to a PhD-yielding scientist working in an elaborate laboratory — there is an option for all ends of the spectrum.

What is Plant Tissue Culture?

When we say plant tissue culture, what we’re really talking about is micropropagation. Micropropagation is a form of cloning plant tissue on a very small scale, and I mean very small. For instance, with micropropagation methods, a clone can be created from just a tiny leaf, bud, or root segment of the mother plant. This varies drastically from traditional cloning methods in which an entire branch of a vegetative plant is required for successful rooting.

Plant tissue culture has been around for decades, originating as a solution for hard-to-germinate orchids. Now, plant tissue culture is the standard for commercial nurseries and many commercial-scale cultivators, ranging from potatoes to flowers to fruit trees.

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Plant Tissue Culture sample in a test tube. Photo courtesy of Swapan Photography.

Micropropagation Vs. Tradition Cloning: Cost Comparison

When comparing micropropagation to traditional cloning or seed propagation, the cost benefits of transitioning to tissue culture become evident. A traditional veg operation requires costly care: a daily dose of water and/or fertilizer, electricity to keep the environmental controls on point, and the substantial space required to house all those vegetative and mother plants. All of those inputs start to add up when we’re talking about a large-scale commercial operation. Cultures, on the other hand, are surprisingly low maintenance. They only need to be divided and transferred to fresh medium every 4-6 weeks — there is no daily watering or caretaking other than casual observation. Micropropagation also allows for the generation of thousands of plants in a very short time period with very small square footage, something that would be impractical with traditional cloning techniques. A mind blowing example, just to put it in perspective, is of a “Day Lily” cultivator who uses plant tissue culture methods to propagate 1,000 day lilies in 30 square feet of shelf space each week. Using conventional methods, one would need a half-acre to produce the same amount of day lilies.

Holly Scroggins lays out a conservative example for a Rhododendron culture in her book Plants from Test Tubes, “Once multiplication gets underway, assuming exponential multiplication (a doubling of material every month), from a single explant you would have 1024 after 10 months, 2048 plants after 11 months, and so on.” And that’s not all, “Of course, you would hope to have more than one successful explant, and you should expect the explants to do more than double each time they are transferred to new cultures.” The rapid multiplication of clones that micropropagation allows outcompetes the speed and number of clones produced using your average cut-and-clone technique.

A full shelf of cannabis tissue culture samples at Cultivar Syndicate. Photo courtesy of Oleg Zharsky.

A full shelf of cannabis tissue culture samples at Cultivar Syndicate. Photo courtesy of Oleg Zharsky.

An Overview of the Micropropagation Process

First, a sterile environment/workspace cannot be emphasized enough. Equally as important is full environmental control: temperature, humidity, and light intensity control are all necessary for successful micropropagation.

Cannabis Tissue Culture lab at Cultivar Syndicate. Photo courtesy of Oleg Zharksy.

Cannabis Tissue Culture lab at Cultivar Syndicate. Photo courtesy of Oleg Zharksy.

Step One begins with the mother plant. The future clones will be an exact replica of the mother plant, so the mother plant should be healthy and exhibiting all desired characteristics. In this first step, a cutting is taken off the mother plant — for micropropagation a cutting can be a fresh shoot, a leaf segment, or a node. To ensure success, a fresh shoot/branch is the best option.

Step Two involves cleaning and disinfecting the cutting, or in tissue culture terms, the “explant.” Any excess or larger leaves are removed from the sample. The explant is rinsed of any contaminants with ethanol, bleach, and water (distilled or rainwater) and then placed into a test tube.

Step Three is the multiplication period for the tiny explants. If properly cared for, one explant will multiply indefinitely.

Step Four is where in vitro rooting begins. “In vitro” translates to “in glass.” This is the stage when the plant material develops small, juvenile shoots.

Step Five is the transplantation and acclimatization stage. This stage hardens off the new sprouts so they are prepped for success.

Pros and Cons

Pros:

Space Saver, much less storage is required to preserve genetics

Provides exact replicas of the mother plant, creates uniform offspring

Sterile Environment reduces risk of pests and disease

Speeds up the breeding / pheno-hunting process

Able to produce an endless amount of plants from one “cutting” (explant).

Minimal daily care

Allows for year-round propagation

Cons:

Steep build-out investment for a large-scale operation

Requires patience, takes more time than traditional cloning if conducted on a small scale

Requires extreme attention to detail

Sterile/controlled environment is necessary

It’s a new beast to master

Veg plants at Cultivar Syndicate. Photo courtesy of Oleg Zharsky.

Veg plants at Cultivar Syndicate. Photo courtesy of Oleg Zharsky.

Tissue Culture on a Budget

If the idea of building out a sterile lab-grade environment is intimidating, as it is to most home-growers, there are a couple different options for experimenting with micropropagation on a budget.  

One option is to buy a tissue culture kit. There are a variety of different kits sold online that range from $150-$300. These kits are definitely more for home-experimentation, but are a great step in the right direction for beginners who aren’t positive they want to commit to building an entire tissue culture lab.

Another option is to build a miniature tissue culture lab with some easy-to-find household materials. A glass or plastic chamber, such as a container you’d buy berries in, works great as an inexpensive greenhouse alternative. Pre-mixed media (nutrient-gel) used to start the cultures can be purchased online. Sterilization is key, but a sanitized table can easily serve as a workspace during the transfer stage of the cultures every 4-6 weeks. With enough patience and determination, anyone can have success with tissue culture!

Is Micropropagation Really an Alternative to Cloning?

Kevin Jodrey, founder of Wonderland Nursery. Photo courtesy of @WonderlandNursery

Kevin Jodrey, founder of Wonderland Nursery. Photo courtesy of @WonderlandNursery

After listening to Kevin Jodrey, owner of Wonderland Nursery, speak at Portland’s Weed Week seminars, I was inspired to incorporate tissue culture into our own operation at TKO Reserve. But is micropropagation practical for commercial-scale growers? Kevin’s answer fluxed between a hesitant ‘yes but no.’

Micropropagation is a standard in the commercial nursery industry — in Kevin’s opinion this is where tissue culture should stay, in a nursery setting. When Wonderland Nursery is fulfilling an order of a couple thousand plants, the only practical way to execute is via micropropagation. However, Kevin doesn’t use micropropagation for his own cannabis farm, which is saying a lot from someone who is an expert in this field.

In his opinion, micropropagation could be an alternative to cloning in the future, with a strong emphasis on the future. But implementing tissue culture labs at every farm is somewhat impractical. When you look at the fruit tree industry, that is not what is happening. Large-scale fruit tree cultivators often source their micropropagated clones from an experienced nursery. As the cannabis industry expands and becomes integrated throughout the states, farms are likely to follow a similar model.

But in the meantime, cannabis farms are establishing personal tissue culture labs left and right. So let’s talk about the logistics of creating your very own lab-grade micropropagation lab.

Commercial-scale Tissue Culture

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Laminar Flow Hood at Cultivar Syndicate. Photo couresy of Oleg Zharsky.

Required Equipment:

  • Laminar Flow Hood
  • HEPA Filter
  • Magnetic stirrer
  • pH measure
  • Glass measuring gradual
  • Culture bottle with lid
  • Dissection tools (Forceps, spatula, scalpel, tweezers, scissors)
  • Conical flasks
  • Refrigerator
  • Microwave
  • Pipette measuring
  • Shaker
  • Aluminum foil
  • Disinfectants (Ethanol, Clorox, Tween 20)
  • Bunsen burner (to sterilize equipment)

The first step of micropropagation process is prepping the media. The media used during the first stage of micropropagation is a nutrient-rich substrate filled with chemical compounds designed for growing cultures, essentially food for the plant tissue. Recipes and techniques are constantly changing as scientists experiment and adapt to what suits plant cultures best.

Pre-mixed medias are available to purchase online, which I recommend. You can always make your own but the recipe calls for a variety of uncommon nutrients. For those who are curious, here’s what goes into the average micropropagation media:

  • Macronutrients: NH4NO3, KNO3, CaCl2.2H2O, MgSO4 7H2O, KH2PO4
  • Gamborg’s B5 Vitamins: Myo-inositol, Nicotinic Acid, Pyridoxine, Thiamine HCI
  • Micronutrients: H3BO3, MnSO4.H2O, ZnSO4.7H2O, KI, Na2MoO4.2H2O, CuSO4.5H2O, CoCl2.6H2O
  • FeNaEDTA
  • Sucrose
  • Agar
  • Coconut water (optional)
  • Distilled Water
  • The pH is then adjusted to 5.8 using hydrochloric acid or sodium hydroxide.
Detail of a cannabis leaf. Photo courtesy of Allie Beckett.

Detail of a cannabis leaf. Photo courtesy of Allie Beckett.

The next step is prepping the explant (plant material). Freshly sprouted nodes, the newest growth on the mother plant, is the best source material because it hasn’t had a chance to be exposed to diseases or pathogens. Once the cutting has been taken, the plant material needs to be heavily disinfected or else we risk the growth of unwanted cultures. To clean, simply place your explant in a test tube with ethanol, swish around for a couple minutes, drain, and repeat the process one more time. Discard the ethanol and rinse your plant material (still in the test tube) with distilled water. Discard the water and leave the plant material in a laminar flow hood to dry.

Once the explant is prepped, it’s time to sterilize the work station and all material with ethanol. This step requires a conical flask and forceps which both need to be sterilized. Once the conical flask is filled with the media, the explant is transferred to the flask and sealed with a cap or covered with aluminum foil.

Flasks need to be kept in a mild-temperature environment with plenty of light. And before you know it, infant seedlings will begin to form in a couple weeks!

After a month or two, depending on the plant, the seedling will be noticeably developed and ready for transplant. Since the seedlings were created in such a sterile, controlled environment, it’s important not to shock them during transplant. A small terrarium or greenhouse environment is recommended as humidity and temperature should not fluctuate for the first week. You can make your own humidity dome with a plastic bag placed over the individual pot — poke a few holes in the bag and spray with water and voila, you have a humidity chamber.

Over the next couple of weeks, slowly harden off the plantlet by gradually exposing it to a more dry and bright environment.

As long as the media is kept fresh and changed every 4-6 weeks, plant genetics can be preserved via plant tissue culture for many years.

Cannabis Tissue Cultures at Cultivar Syndicate. Photo courtesy of Oleg Zharsky.

Cannabis Tissue Cultures at Cultivar Syndicate. Photo courtesy of Oleg Zharsky.

Let the Micropropagation Begin!

That’s a wrap on our introduction to plant tissue culture and micropropagation. As you can see, there are hundreds of different methods and applications for plant tissue culture, the possibilities are truly endless. I hope this Grow Guide shows you that getting started with plant tissue culture isn’t as intimidating as it’s made out to be. I’d love to hear your stories in the comment section below!

About Author

Allie is a NW-based content curator for Marijuana.com and an organic farmer at TKO Reserve. She has been a professional in the marijuana industry since she was 18 years old, spending the first five years of her career working for Dope Magazine as lead photographer. Allie has worked on mainstream projects such as Idiot's Guide: Growing Marijuana, Branding Bud: The Consumerization of Cannabis and her own self-published book, As The Grass Grows.

4 Comments

  1. Tissue culture can serve as a viable, cost effective method to preserve landrace and other rare strains for future breeders and researchers.

  2. Please contact me when you get this. I am presenting Cannabis Tissue Culture to the scientists of In Vitro in June and would like to share the accomplishments of your lab.

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