Learn about how to decide whether seed, clone, or tissue culture is right for you with Dr. Allison Justice's guest column in the January 2021 issue of Cannabis Business Times:
In the ornamental and produce industries, we refer to genetic starting material as “young plants.” This includes seed, clone, and tissue production, all three options are commonly used, all for different reasons. Each type of young plant has its purpose and place for use. For plants with seed lines that contain high heterozygosity (lots of genetic variability) or do not produce seed at all, clonal production is the only way to produce a consistent crop. For plants which are homozygous with stable genetics, seed is an easy, inexpensive choice. Tissue culture is a method of reproducing plants clonally in sterile culture. In cannabis production, all three options are commonly used, all for different reasons.
Seed is a great choice for cannabis propagation. They are readily available, inexpensive, and come with a low risk of carrying pest or disease. That said, for most cannabis growers, plants grown from seed are not consistent enough to grow vegetatively and flower. Consumers expect consistency in product, so variation is avoided.
Starting from seed usually means cannabis growers must go through a “pheno-hunt” prior to incorporating the seed into commercial production. A pheno-hunt is a technique of germinating seedlings, taking matched clones from each seedling, flowering each seedling and then choosing the phenotype with the most desirable traits, from chemical composition to pest and disease resistance and physical attributes. From here, the seedling becomes a clonal plant, which can then go into commercial production or into tissue culture. This is a great process to find a unique phenotype for an operation but can take a significant amount of time to determine the right selection.
Seed can carry diseases such as Fusarium spp. and tobacco mosaic virus internally and on the coat, so it’s important to surface sterilize them using sodium hypochlorite (1% to 5%, testing a small batch first) to prevent spread.
Also important to remember is that cannabis is monoecious (male and female flowers grow on separate plants). This means if regular seed (non-feminized) is purchased for cannabinoid production, about half of those seeds will be male and useless (unless the grower is conducting breeding work). To save time and space, seedlings can be genetically tested when leaves are about the size of a quarter. Depending on volume, this can cost $5 to $15 per test but can save a lot of wasted space in the long run.
The other option is to buy feminized seed treated with chemicals that reduce ethylene production, causing female plants to produce male flowers. These self-pollinating plants produce all female seed, but many times true males or hermaphrodites will appear. This is a risk, as males or hermaphrodites could spread pollen in the room, pollinating female flowers and forcing those plants to direct energy to seed production instead of cannabinoid production.
Many cannabis growers start their crops from clones. Clones provide a true replication of the desired phenotype and yield a consistent crop. Compared to many other plant species, cannabis is easy and fast to propagate.
Clones originate from internally pheno-hunted seed or bought from a cannabis nursery. Many growers do not have space to conduct a scaled pheno-hunt, so buying from a nursery can be a fast way to incorporate a phenotype that has already undergone a rigorous selection process. This saves time, space and money. Ordinarily, pheno-hunting can take at least three months to select a specific phenotype and ramp up numbers for production. If clones are bought, they can be incorporated into production after quarantining. Additionally, the flowering space required to pheno-hunt could equate to lost revenue if the seed lineage produces poor quality flower or males/hermaphrodites.
There is risk of bringing in diseases or pests on clones. Always ask the nursery about their Integrated Pest Management (IPM) protocols and how often they send in their stock for pathogen testing. Having a quarantine area for new incoming genetics also is crucial.
Plant health issues, especially those caused by insects, that may not be immediately obvious could manifest during the quarantine period and before infecting the crop.
The origins of plant tissue culture can be traced to Austrian botanist Gottlieb Haberlandt, who studied tissue culture at the beginning of the 20th century. The 1940s to 1960s were an exciting time in understanding plant behavior and technique in cell culture, but it was not until the 1990s that it truly found a place in commercial horticulture. Tissue culture provided a way for commercialization of certain plant species, such as orchids, that otherwise could not occur. Tissue culture of the root tips at that time was the only way to produce a virus-free plant.
Tissue culture is becoming more widely used in the cannabis industry. (Home kits are even sold at hydroponics shops.) However, before implementing this technology in a cultivation operation, it is imperative to understand the science. The industry is weeding out the businesses looking to take advantage of customers with false product claims. But there is still misleading information out there of which growers and businesses considering purchasing tissue cultured plants for their facility, refining their genetics, or looking for a genetic banking option need to be aware.
UNDERSTANDING THE BASICS OF TISSUE CULTURE
Tissue culture is the culture of plant cells (tissues or organs) in an aseptic (sterile) and environmentally controlled vessel. Light is pro- vided for photosynthesis, and the plants also are provided with carbohydrates (sugars).Within the umbrella definition of tissue culture is micropropagation (MP). Micropropagation is the tissue culture technique of vegetative plant multiplication. Put simply, micropropagation can be thought of as sterile cloning in a small, controlled vessel.
So why would someone go to this length when cloning cannabis can be so easy? Because having clean plants is imperative! Pesticide use in cannabis is highly regulated, so choices for control are minimal. The ability to start with a clean slate will save time, money, prevent loss, and yield higher quality product. This is a great way for breeders to move their germplasm repository out of the growing facility and “bank” plants in a safe, secure, and sterile area, which can be used later. There are many other benefits to MP, especially for growers who do not have an in-house laboratory.
SCREENING AND ENSURING QUALITY
There are four stages to micropropagation (see sidebar above). In Stage I, the growing points are harvested as explants to disinfect and stabilize. The first step is surface sterilization, which, in-house, is usually done by an explant dip into a bleach or alcohol mixture. This eliminates insects and surface pathogens.
One tissue cultured sample can cost five to 10 times more than seed, so growers must be certain they are getting what they pay for. A proper TC facility should be screening for eight or more viruses and viroids, and a list of well over 10 for other fungi and bacteria in Stage I. If proper screening is not done within Stage I, the service is no more valuable than an in-house dip.
To ensure MP is more valuable than an in-house dip, it’s important for growers to ask labs: What are the steps used to ensure any systemic pathogen is removed? Some tissue culture companies prefer to use antibiotics and fungicides to ensure a clean explant, but potential drawbacks to this procedure include chemical resistance, lingering pesticide in the plant material, and pollution from excess chemical use in the laboratory. Other labs will grow and transfer plant material to new vessels until the pathogen no longer expresses itself on the medium. The latter option can take longer, for obvious reasons.
Regardless of the cleaning method, the cutting is not 100% guaranteed to be clean until the plantlet is sent to a pathology clinic and screened. Growers should ask the laboratory not only if it screens, but again for how many pathogens. If it only can provide two or three, with one of those being botrytis, growers should dig deeper to find out why. If the lab claims it cannot screen in-house, growers can suggest a pathology clinic the lab can work with to guarantee clean material.
One pathogen that is commonly present and should be screened for is Fusarium spp. This fungal disease is a silent killer that only shows itself in the highest of stress conditions and usually late into flower. Traditionally, the symptoms were associated with poor growing techniques or improper fertilization, but now, with testing, growers can manage this disease properly.
Understanding the stage and type of plant growers are receiving back from the laboratory also is important to know when making a tissue culture lab selection. Just because a company is marketing MP liners does not mean the liner itself was in tissue culture; the liner could be a clone from a plant that was in tissue culture months ago. Many companies will produce mother stock from such MP plants that are used for a clonal propagation program. This can be a great practice, especially when in-house stock is exchanged at regular intervals. That said, once a plant enters a greenhouse, it is no longer sterile. Without proper IPM, this once-clean plant can become infected, making the MP step useless.
Labs that provide Stage III plants have two plant options they can deliver: plants that have been rooted within or outside the sterile container. In most cases, both scenarios are safe, yet growers should still ask for details. For example, if roots are established outside the sterile container in a greenhouse under mist, this could be a source of new disease inoculation.
Genetic banking via tissue culture is an option to store and preserve germplasm for long periods of time for clonal plants. There are many reasons a grower may want to enter genetics into in vitro banking. Square footage in a permitted grow facility is expensive, and vegetative space can be limited. This method allows growers to store genetics they may wish to breed or simply reintroduce into commercial rotation later. This also allows a company to hold on to valuable genetics without needing space for a mother plant.
Genetic banking also can serve as clean stock back up. For example, imagine a grower’s three top-selling genetics that are always in rotation suddenly become diseased and must be destroyed. If there are already clean plants in MP, that grower is a step ahead. On the flip side, if that grower waits to undergo MP until they realize their plants are diseased, getting a plant to a point of reintroduction can take upwards of six months if there is low disease pressure. Also, note that reintroducing an MP plant from long-term banking can take a couple months.
In cannabis, everything starts from seed, but it is up to each grower to decide the best business decision for what happens after that seed has popped.
- Thorpe A. (2006) History of Plant Tissue Culture. In: Loyola-Vargas V.M., Vázquez-Flota F. (eds) Plant Cell Culture Protocols. Methods in Molecular Biology™, vol 318. Humana Press. https://doi.org/10.1385/1-59259-959-1:009.
- Chandana, C., Kumari Nagaveni, H. C., Heena, M. S., Shashikala, S. K., & Lakshmana, D. (2018). Role of plant tissue culture in micropropagation, secondary metabolites production and conservation of some endangered medicinal crops. J. Pharmacogn. Phytochem, 246-251.