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Applications for Tissue Culture in Cannabis Growing: Part 2

By Aaron G. Biros
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In the first part of this series, we introduced Dr. Hope Jones, who took her experience in tissue culture from NASA and brought it to the cannabis industry and C4 Laboratories. We discussed some of the essential concepts behind tissue culture and defined a few basic terms like micropropagation, totipotency, explants and cloning. Now let’s get into some of the issues with cloning from mother plants and the advantages that come with using tissue culture for propagating and cultivating cannabis.

Time & Resources

Dr. Hope Jones, chief scientific officer at C4 Labs

Taking cuttings from mother plants is arguably the most popular method of propagating cannabis plants. It is a process that requires significant real estate, resources and labor. “Moms can take up a great deal of space that is not contributing directly to production,” says Dr. Jones. “I know from experience that scaling up production and/or adding new strains to the production line requires significant time and resources to raise and maintain new healthy and productive mother plants.” Each mother plant produces a limited number of clones per harvest period and over the course of her life cycle.

By using tissue culture, a cultivator can generate an almost infinite number of clones from one plant cutting. With so many growers calculating their costs-per-square-foot, micropropagation is an effective tool to save space, labor and time, thus increasing profit margins. “Just to put it in perspective: Holly Scoggins’ book Plants From Test Tubes, cites a Day Lily cultivator who uses micropropagation to produce 1,000 plants in 30 square feet of shelf space each week,” says Dr. Jones. “Using conventional methods, one would need a half-acre to produce the same amount of plants.” Cultivators can produce a much greater number of plants-per-square-foot by using micropropagation effectively.

Damage from whiteflies, thrips and powdery mildew is all visible on this sick plant.

Early Health & Vigor

Most tissue culture methods use sterilized vessels that contain sugar-rich media to support growth of plantlets before they can photosynthesize on their own. “The media is prepped, poured into vessels, and placed in an autoclave (or pressure cooker) where it is subjected to high temps and pressure to achieve proper sterility.”

The sterile environment and rich growth media supplies plantlets with an abundance of everything they need. “When plantlets emerge from culture, they are pathogen-free, with a stockpile of food and nutrient reserves that support rapid growth and vigor, superior to conventional cuttings,” says Dr. Jones.

Stress & Disease

As any grower knows, mother plants can sometimes experience stress and disease. This might come in the form under or over-watering, heat stress, spider mites, whiteflies, mold and viruses. “Any stress or infection that a mother plant is subjected too can impact progeny health and productivity in a couple of ways,” says Dr. Jones.

Powdery mildew starts with white/grey spots seen on the upper leaves surface
Tobacco Mosaic Virus symptoms can include tip curling, blotching of leaf mosaic patterning, and stunting.

For example, diseases like powdery mildew and tobacco mosaic virus are often systemic, meaning that pathogens have spread to almost every tissue in the plant. Once infected, it is impossible to completely eliminate pathogens from tissues. Therefore any cuttings made from a diseased mother plant, even if they look perfectly healthy, will also be infected and can eventually present disease symptoms like reduced productivity and/or plant death, according to Dr. Jones.

How does tissue culture get around this problem? Remember that explants (small tissue samples used as starting material) can be extracted from any part of the plant. Meristematic cells in shoot tips and leaves are the source of new plant growth. Dr. Jones explains that these cells, and the first set of primordial leaves are not connected directly to the vascular tissue, the plant’s transport system by which pathogens spread. Therefore, meristematic cells tend to be disease-free, whatever the condition of the mother. It takes a sharp blade, a dissecting microscope, and a lot of experience to learn, but as Dr. Jones explains, “harvesting explants from meristems is a routine micropropagation technique used by ‘Big Horticulture.’ One example is the strawberry. Viruses and pathogens are so prevalent that the strawberry industry must use meristematic culture to ensure pathogen free progeny.”

Epigenetics

Now let’s talk about epigenetics. We know that plants don’t have the option of physically moving away from stress or predation. Instead, they have evolved sophisticated ways of changing their own biology to adapt to and/or protect themselves. “Consider what happens to a mom exposed to a pathogen. The infected plant will start expressing (turning on) genes and making proteins that contribute to pathogen resistance,” says Dr. Jones. “These changes to gene expression are partly regulated by epigenetic modifications, chemical changes to DNA that increase or decrease the likelihood a cell will express a particular gene, but that do not actually modify the gene itself. Like annotations to a piece of music, epigenetic modifications don’t change the notes but rather how loud or soft, quickly or slowly the notes are played.”

There are more than 1,000 different viruses and mixed infections are very common

This is where it gets interesting. “Epigenetic modifications can be systemic and long lived. Plants infected by a pathogen or stressed by drought will present widespread epigenetic modifications to their DNA,” says Dr. Jones. “For an annual plant like cannabis, those modifications are relatively permanent. Thus a cutting from a mom having drought or pathogen adapted epigenetic programming will inherit that modified DNA and behave as if it were experiencing that stress, whether present or not.”

In the wild, this adaptability is critical for plant survival and reproduction, but to a grower, this is a less-than-ideal scenario. “The epigenetic modifications allowed the mother to tolerate the stress, which is great from the perspective of survival and fitness, but it comes at a cost. Some of the finite energy and resources that usually support plant growth and reproduction are instead channeled to stress response,” says Dr. Jones. This trade off results in reduction in overall plant yield and quality. “Those epigenetic changes result in a new phenotype for that mother,” says Dr. Jones. “All cuttings from her will reflect the new phenotype. This is one major mechanism underlying what many in the cannabis industry (incorrectly) call ‘genetic drift,’ or the loss of vigor over successive clonal generations.”

This is again where tissue culture can be such a game changer. The process of dedifferentiation, as explained in part 1 of this series, can rejuvenate a “tired” mother plant by inducing a kind of reboot– clearing accumulated epigenetic modifications that negatively impact progeny vigor and productivity. In the third part of this series, we will discuss the five stages of micropropagation, detailing the process of how you can grow plantlets in tissue culture. Stay tuned for more!

Applications for Tissue Culture in Cannabis Growing: Part 1

By Aaron G. Biros
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Dr. Hope Jones, chief scientific officer of C4 Laboratories, believes there are a number of opportunities for cannabis growers to scale their cultivation up with micropropagation. In her presentation at the CannaGrow conference recently, Dr. Jones discussed the applications and advantages of tissue culture techniques in cannabis growing.

Dr. Hope Jones, chief scientific officer at C4 Labs

Dr. Jones’ work in large-scale plant production led her to the University of Arizona Controlled Environment Agriculture Center (CEAC) where she worked to propagate a particularly difficult plant to grow- a native orchid species- using tissue culture techniques. With that experience in tissue culture, hydroponics and controlled environments, she took a position at the Kennedy Space Center working for NASA where she developed technologies and protocols to grow crops for space missions. “I started with strawberry TC [tissue culture], because of the shelf life & weight compared with potted plants, plus you can’t really ‘water’ plants in space- at least not in the traditional way,” says Dr. Jones. “Strawberries pack a lot of antioxidants. Foods high in antioxidants, I argued, could boost internal protection of astronauts from high levels of cosmic radiation that they are exposed to in space.” That research led to a focus on cancer biology and a Ph.D. in molecular & cellular biology and plant sciences, culminating in her introduction to the cannabis industry and now with C4 Labs in Arizona.

Working with tissue culture since 2003, Dr. Jones is familiar with this technology that is fairly new to cannabis, but has been around for decades now and is widely used in the horticulture industry today. For example, Phytelligence is an agricultural biotechnology company using genetic analysis and tissue culture to help food crop growers increase speed to harvest, screen for diseases, store genetic material and secure intellectual property. “Big horticulture does this very well,” says Dr. Jones. “There are many companies generating millions of clones per year.” The Department of Plant Sciences Pomology Program at the Davis campus of the University of California uses tissue culture with the Foundation Plant Services (FPS) to eliminate viruses and pathogens, while breeding unique cultivars of strawberries.

A large tissue culture facility run in the Sacramento area that produces millions of nut and fruit trees clones a year.

First, let’s define some terms. Tissue culture is a propagation tool where the cultivator would grow tissue or cells outside of the plant itself, commonly referred to as micropropagation. “Micropropagation produces new plants via the cloning of plant tissue samples on a very small scale, and I mean very small,” says Dr. Jones. “While the tissue used in micropropagation is small, the scale of production can be huge.” Micropropagation allows a cultivator to grow a clone from just a leaf, bud, root segment or even just a few cells collected from a mother plant, according to Dr. Jones.

The science behind growing plants from just a few cells relies on a characteristic of plant cells called totipotency. “Totipotency refers to a cell’s ability to divide and differentiate, eventually regenerating a whole new organism,” says Dr. Jones. “Plant cells are unique in that fully differentiated, specialized cells can be induced to dedifferentiate, reverting back to a ‘stem cell’-like state, capable of developing into any cell type.”

Cannabis growers already utilize the properties of totipotency in cloning, according to Dr. Jones. “When cloning from a mother plant, stem cuttings are taken from the mother, dipped into rooting hormone and two to five days later healthy roots show up,” says Dr. Jones. “That stem tissue dedifferentiates and specializes into new root cells. In this case, we humans helped the process of totipotency and dedifferentiation along using a rooting hormone to ‘steer’ the type of growth needed.” Dr. Jones is helping cannabis growers use tissue culture as a new way to generate clones, instead of or in addition to using mother plants.

With cannabis micropropagation, the same principles still apply, just on a much smaller scale and with greater precision. “In this case, very small tissue samples (called explants) are sterilized and placed into specialized media vessels containing food, nutrients, and hormones,” says Dr. Jones. “Just like with cuttings, the hormones in the TC media induce specific types of growth over time, helping to steer explant growth to form all the organs necessary to regenerate a whole new plant.”

Having existed for decades, but still so new to cannabis, tissue culture is an effective propagation tool for advanced breeders or growers looking to scale up. In the next part of this series, we will discuss some of issues with mother plants and advantages of tissue culture to consider. In Part 2 we will delve into topics like sterility, genetic reboot, viral infection and pathogen protection.

Understanding Dissolved Oxygen in Cannabis Cultivation

By Aaron G. Biros
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Oxygen plays an integral role in plant photosynthesis, respiration and transpiration. Photosynthesis requires water from the roots making its way up the plant via capillary action, which is where oxygen’s job comes in. For both water and nutrient uptake, oxygen levels at the root tips and hairs is a controlling input. A plant converts sugar from photosynthesis to ATP in the respiration process, where oxygen is delivered from the root system to the leaf and plays a direct role in the process.

Charlie Hayes has a degree in biochemistry and spent the past 17 years researching and designing water treatment processes to improve plant health. Hayes is a biochemist and owner of Advanced Treatment Technologies, a water treatment solutions provider. In a presentation at the CannaGrow conference, Hayes discussed the various benefits of dissolved oxygen throughout the cultivation process. We sat down with Hayes to learn about the science behind improving cannabis plant production via dissolved oxygen.

In transpiration, water evaporates from a plant’s leaves via the stomata and creates a ‘transpirational pull,’ drawing water, oxygen and nutrients from the soil or other growing medium. That process helps cool the plant down, changes osmotic pressure in cells and enables a flow of water and nutrients up from the root system, according to Hayes.

Charlie Hayes, biochemist and owner of Advanced Treatment Technologies

Roots in an oxygen-rich environment can absorb nutrients more effectively. “The metabolic energy required for nutrient uptake come from root respiration using oxygen,” says Hayes. “Using high levels of oxygen can ensure more root mass, more fine root hairs and healthy root tips.” A majority of water in the plant is taken up by the fine root hairs and requires a lot of energy, and thus oxygen, to produce new cells.

So what happens if you don’t have enough oxygen in your root system? Hayes says that can reduce water and nutrient uptake, reduce root and overall plant growth, induce wilting (even outside of heat stress) in heat stress and reduce the overall photosynthesis and glucose transfer capabilities of the plant. Lower levels of dissolved oxygen also significantly reduce transpiration in the plant. Another effect that oxygen-deprived root systems can have is the production of ethylene, which can cause cells to collapse and make them more susceptible to disease. He says if you are having issues with unhealthy root systems, increasing the oxygen levels around the root system can improve root health. “Oxygen starved root tips can lead to a calcium shortage in the shoot,” says Hayes. “That calcium shortage is a common issue with a lack of oxygen, but in an oxygen-deprived environment, anaerobic organisms can attack the root system, which could present bigger problems.”

So how much dissolved oxygen do you need in the root system and how do you achieve that desired level? Hayes says the first step is getting a dissolved oxygen meter and probe to measure your baseline. The typical dissolved oxygen probe can detect from 20 up to 50 ppm and up to 500% saturation. That is a critical first step and tool in understanding dissolved oxygen in the root system. Another important tool to have is an oxidation-reduction potential meter (ORP meter), which indicates the level of residual oxidizer left in the water.

Their treatment system includes check valves that are OSHA and fire code-compliant.

Citing research and experience from his previous work, he says that health and production improvements in cannabis plateau at the 40-45 parts-per-million (ppm) of dissolved oxygen in the root zone. But to achieve those levels, growers need to start with an even higher level of dissolved oxygen in a treatment system to deliver that 40-45 ppm to the roots. “Let’s say for example with 3 ppm of oxygen in the root tissue and 6ppm of oxygen in the surrounding soil or growing medium, higher concentrations outside of the tissue would help drive absorption for the root system membrane,” says Hayes.

Reaching that 40-45 ppm range can be difficult however and there are a couple methods of delivering dissolved oxygen. The most typical method is aeration of water using bubbling or injecting air into the water. This method has some unexpected ramifications though. Oxygen is only one of many gasses in air and those other gasses can be much more soluble in water. Paying attention to Henry’s Law is important here. Henry’s Law essentially means that the solubility of gasses is controlled by temperature, pressure and concentration. For example, Hayes says carbon dioxide is up to twenty times more soluble than oxygen. That means the longer you aerate water, the higher concentration of carbon dioxide and lower concentration of oxygen over time.

Another popular method of oxidizing water is chemically. Some growers might use hydrogen peroxide to add dissolved oxygen to a water-based solution, but that can create a certain level of phytotoxicity that could be bad for root health.

Using ozone, Hayes says, is by far the most effective method of getting dissolved oxygen in water, (because it is 12 ½ times more soluble than oxygen). But just using an ozone generator will not effectively deliver dissolved oxygen at the target levels to the root system. In order to use ozone properly, you need a treatment system that can handle a high enough concentration of ozone, mix it properly and hold it in the solution, says Hayes. “Ozone is an inherently unstable molecule, with a half-life of 15 minutes and even down to 3-5 minutes, which is when it converts to dissolved oxygen,” says Hayes. Using a patented control vessel, Hayes can use a counter-current, counter-rotational liquid vortex to mix the solution under pressure after leaving a vacuum. Their system can produce two necessary tools for growers: highly ozonized water, which can be sent through the irrigation system to effectively destroy microorganisms and resident biofilms, and water with high levels of dissolved oxygen for use in the root system.