Cannabis Plant Tissue Culture by THC Design
Another method of cloning is plant tissue culture, also known as micropropagation, or in-vitro propagation, from the Latin, meaning “in glass”. To put it simply, tissue culture is essentially sterile cloning. Multiplying cells and plants in an exponential fashion in a sterile environment.
Plant tissue culture has been around for a long time. Experimental plant tissue culture first began in 1898, but it didn’t come into widespread use until the 1950s, in the commercial orchid industry. Of course, due to the historic legal hurdles, research in the field of cannabis tissue culture is still in its infancy.
Plant Tissue Culture Uses
- Increased Speed of Growth
- Genetic Rejuvenation
- Genetic (Germplasm) Preservation
- Virus Elimination
- Genetic Engineering
Some plant tissue culture techniques are more complex than others, and some methods may seem overwhelming or out of reach at first to the beginner grower. Currently at THC Design, we primarily use only the most basic, non-GMO plant tissue culture techniques as a method for cloning and for eliminating pests and disease.
As we gain greater understanding of the full spectrum of cannabinoids and terpenes found in cannabis, we’ll be able to “dial-in” the genetics of our strains to enhance certain traits or increase the potency of select cannabinoids or terpenes. Designer, bespoke cannabis.
The Plant tissue culture process:
Sterilization of equipment and work space is critical throughout.
Initiation Phase (1 to 2 weeks)
Pathogens will typically grow in the media or express themselves in the plantlets within the first two weeks. By observing the traits of the plantlets, having the plant tissue tested at a third-party commercial viral testing lab, or performing a strip test, we can determine if elimination of the pathogen was successful or not. The successfully cleaned and healthy plantlets are then divided, added to a multiplication media and then moved to larger containers. During this stage, the plantlets must be divided and transferred to fresh nutrient media regularly. We typically get 5 generations of multiplication before the genetics start to degrade.
- Increased Speed of Growth
- Genetic Rejuvenation
Initiation Phase (1 to 2 weeks)
- Turn on the laminar flow hood, and allow it to run for at least 30 minutes before attempting the procedure. If you do not have a laminar flow hood, use an alcohol lamp/ burner and work as closely as possible to the flame.
- Sterilize the laminar flow hood with 70% Ethanol. Do not spray directly into the back of the hood as this can cause damage to the HEPA filter.
- Turn on your bead sterilizer. It will take at least 20 min to warm up to temperature but this may depend on the manufacturer.
- Sterilize a pack of paper in an autoclavable pouch, the vessels you will eventually place the plants in, several liters of water, and your tween 20 in your autoclave. Make sure to run liquids on a liquid/ slow exhaust cycle (never fill containers more than 50% full) and the paper on a dry cycle.
- Prepare a batch of media as follows:
- Obtain a container that is large enough to hold the amount of media you want to make.
- Fill the container with the purest water available. Distilled, RO, or DI water is ideal. If you are making 1L, add about 90% of your total volume, or 900 mL.
- Add vegetative cannabis nutrients until you reach 1.5 EC
- Add 30 g/L of sugar.
- pH the solution to 5.7 with 1.0 M HCl or NaOH
- Add 6 g/L agar (note- agar will not mix evenly unless you heat the solution on a hot plate. If you are dividing your media into multiple bottles, it is best to add the appropriate amount of agar to the individual bottles- e.g. for a 1L bottle with 500mL media in it, add 3.5 g Agar to the bottle).
- Pour your media into your autoclavable bottles. NEVER completely fill the bottle as it will overflow during autoclaving. Half full is a good target.
- Autoclave on a liquid cycle with 15 min sterilization time.
- When the cycle is done, remove the media (note autoclave is dangerous- see safety guidelines).
- Pour the media into your sterilized vessels, at about 50 mL, or 1-2in depth, per vessel.
- Prepare a solution of 0.5% bleach (15% v/v) and 0.1% sterile Tween 20 (1g or 1mL per 1L).
- Most bleach is 2.75%, so add 181.81 mL per 1L.
- Fill a container with a small amount of water, take cuts of your cannabis plant material, and place them in water. You can cut an entire branch off, then separate it into 2-4 inch segments that each contain one node. The new plant material will grow from the node. Ensure plant material is healthy.
- If storing cuttings for more than 1 hour before initiation begins, place the cutting in a cooler with ice. A wet paper towel may be wrapped around any exposed cuts in the tissue. In general try to get your cuts into water as soon as possible.
- Obtain a sealable sterilized vessel or container. Fill it about 80% full of your bleach/ tween solution. Add the cannabis segments to the container and seal tightly.
- Shake/ swirl gently for 20 min. Shake vigorously for 20 seconds at the end of the 20 minute period.
- The shaker table may be used for shaking.
- Transfer explants to another sterilized vessel filled
80% full with sterilized water, and shake/ swirl gently for 5 minutes
- Repeat this step another two times.
- Using sterile forceps, transfer explants to a sterile piece of paper to soak up any excess water, then onto the media vessels you prepared, one explant per vessel.
- The plant should be placed into the media upright so that it is supported, but not all the way down into the bottom of the vessel.
- Place the vessel on a grow rack at 25 +/- 2°C under 18 hour photoperiod for at least 3 weeks.
We are proud to be named to the Leaflink list for the 2nd year in a row. THC Design Estate Eighths ranked among “Best Selling Flower and Pre-Rolls” and we were commended for our Diversity in Leadership. You can view the rest of the winners here.
Another method of cloning is plant tissue culture, also known as micropropagation, or in-vitro propagation, fromthe Latin, meaning “in glass”.
Cannadabis: tissue culture and the future of cannabis cultivation
Cannadabis Medical INC is dedicated to the organic and holistic production of cannabis medicine and related technology.
Cannadabis Medical INC they intend to create a healthier and more consciously aware environment for the cannabis industry, and its participants, to thrive in.
Did you know that Cannadabis are Partners with us? Discover their featured Partner Page about a healthier, environmentally conscious cannabis industry.
The company is a family run company that was founded in Humboldt, Saskatchewan.
Founders, Alexander Calkins, BSc and Markus Li, P.Chem, MBA, are personally and emotionally invested in the science of cannabis. They each have family members that are dealing with incurable ailments, complications of which can often become fatal.
In the search for natural products that will improve the quality and longevity of life, the founders began working with cannabis. While there is no likelihood of a cure, the symptom management has been very positive for their family members. After witnessing the improvements, Cannadabis founders – Calkins and Li, have dedicated themselves to furthering the medical cannabis movement.
Founders, Alexander Calkins, BSc and Markus Li, P.Chem, MBA.
Calkins and Li both have backgrounds in technical science and business. They are experienced cultivators and have a strong understanding of energy systems (practically essential for a power-hungry industry), process automation, and large-scale development.
Their familiarity with multi-industry supply chains has leveraged them into a cannabis development that is simultaneously high-tech, old school, and simple.
Through observation of established global industries, Cannadabis is building a multi-faceted business model based on sustainable practices, a strong genetics portfolio, disruptive technologies, hyper-specialisation, and holistic production.
Driven by a passion to help others in need, Calkins and Li took it upon themselves to bring their methods and expertise to the cannabis world. They recognise and praise the patient independence that medical cannabis can provide.
While they champion the practice of homegrown medicine, they have obligated themselves to providing the safest and highest quality medical products to those who are unable to grow for themselves.
Once Cannadabis has perfected its organic growing system, they will build and operate all future cultivation sites according to (EU) GMP and ISO:9001 2015 standards. By adopting these standards, Cannadabis will have the ability to share their cultivated passion with the world.
To meet the sanitary requirements of GMP and processing limitations of an organic certification, Cannadabis will be using a combination of reactive oxygen, electrolysed water, and radio frequency pasteurisation technologies.
Organic and sustainable practice
Being a medically focused company, Cannadabis recognises that medical consumers have turned to cannabis because they are looking for natural remedies and are becoming increasingly weary of synthetic medicines.
For Cannadabis, producing medical cannabis using anything other than organic methods would transgress the fundamental sentiment that drives the global, medical movement. That is why Cannadabis is committed to attaining internationally recognised organic certifications on expanded production.
The company’s flagship facility is intended to be an R&D focused proving ground for state-of-the-art organic cultivation methods. Cannadabis currently uses an inhouse blended soil, made only with organic ingredients. Their living soil has the benefit of creating terpene dense medicine, reducing cost, and simplifying processes.
With all the nutrients available in the soil, the plants require only water from transplant to harvest. Additionally, the growing medium and all organic waste can be recycled through vermicomposting, further reducing long term costs and needless waste.
Cannadabis will adopt various technologies to reduce energy demand and environmental impact. In addition to using LEDs and solar panels, Cannadabis will use combined heat and power (CHP) (or cooling combined heat power (CCHP)) at their cultivation facilities. CHP units burn natural gas to generate power and the waste heat is used to heat water and the workspace. CHPs are quickly becoming popular for reducing carbon emissions. In certain applications, CHP’s reduce carbon emissions by 30-40%, compared to when power is taken from the grid.
Cannadabis will also divert the combustion CO2 into the growing space. CO2 supplementing supercharges growth naturally, increasing yield by 30-60%, and further reducing the carbon emissions from power generation. In the future, expanded cultivations may integrate pyrolysis of waste biomass, which will supply power and nutrient dense biochar to the living soil.
Cannadabis is aspiring to build a unique indoor growing system that uses a combination of solar power, water recycling, CHP (CCHP), pyrolysis, CO2 supplementation and vermicompost to create a no waste, carbon neutral, minimal input, self-regenerating nutrient, off grid, medical grade, organic, indoor cultivation.
Calkins and Li hope to validate the system and then apply the techniques to food cultivation; this type of system could revolutionise the food production in remote locations, like the northern territories, Alaska and would deliver food supply independence to small communities or reservations. Where biomass is abundant, this system would produce all year, requires only labour as inputs, self-generate power off-grid, and would also be carbon ‘negative’ over extended time frames.
Tissue culture science
On their path to improving growing efficiency, Cannadabis has developed proprietary tissue culture methods specifically for cannabis. These methods are based upon the decades old horticultural practice that has been essential for the sterile propagation of ornamental and food cultivars; non seed propagation.
Developing an inhouse tissue culture system has the following benefits: 1
Tissue culture revitalises cultivars and produces more vigorous plants
• Regeneration from meristem rids systemic disease;
• Propagation is significantly more efficient;
• Starting with 100 traditional cuttings; able to produce 70,000 annual clones;
• Start with 200 tissue culture vials; produce 2 million annual clones;
• Uses 1/10 the space of traditional cloning;
• Per square foot, tissue culturing is >100x more efficient; and
• Two million annual clones could be produced in less than 3000 square feet.
Better preservation of genetics
• 1000 mother cultivars could be stored inside a refrigerator with no care or maintenance for months, sometimes over a year; and
• Pest invasion would not affect mother cultures (many cultivators without tissue culture have lost their entire genetic inventory to viruses and fungi).
Cannadabis will be sharing its tissue culture methods with industry members who want to stay one step ahead of pests and systemic disease. Following more development, they will also be making their organic formulations available.
Having collected and grown a large variety of cultivars, both through seed and clone, the Cannadabis founders have noticed a distinct lack of quality in the genetics market. Over time, most of the popular cultivars of the world have been slowly degraded by deleterious breeding practices like selfing (feminising), backcrossing, and poor mother plant maintenance – which promotes genetic drift.
The current genetics market is rife with ‘breeders’ that take prized clones and spray them with colloidal silver to produce feminised seed, or they are crossed onto their own cultivars and backcrossed until stable seed is produced.
While these name sake creations may capture some of the qualities of the original strain, like trichome density or terpene profile, the progeny will lack the genetic diversity needed to produce healthy plants. Often, these weakened strains have reduced yield, potency, and pest resistance. In response to this, Cannadabis has focused on breeding their own high yield, high potency, flavour dense strains for commercial production.
The Cannadabis team is eager to unveil their propriety strains to the domestic and international medical markets. Over the past few years, the founders have started breeding their own cultivars. Currently, the team has focused on a selection of stabilised true breeds (landrace or F5+) for creating original F1 breeds.
Where the F1 generation is created by breeding male and female plants that are distinctly unique from each other; traditional F1s are created by crossing landrace indicas with landrace sativas.
These crosses need to be done with highly stable and uniquely different parents to produce a true F1 progeny that has abundant hybrid vigour. A plant with true hybrid vigour will typically have higher potency, increased pest resistance, and a higher yield than both parent plants; on average yield can be as high as 20% more than either parent.
Due to the nature of the F1 progeny, very few breeders release true F1 seeds. If highly stable progenitors are not used, the seedstock will be incredibly variable, which is unfavourable for consumers, who typically want consistency in their seed. However, as commercial cultivators, Cannadabis believes that F1 hybrids are essential for producing at large scale. The breeding and phenotyping can be a long and arduous process, the fruits of labour are not without commercial benefit.
Building upon the tissue culture and breeding practices, Cannadabis is quickly developing polyploidisation methods for creating ultra-premium cultivars. Polyploidisation is another common horticultural practice that Cannadabis expects to apply to their cannabis breeding projects.
Polyploidisation is a naturally occurring mechanism where the chromosomes of the plant cells become doubled within the same nucleus. This mechanism has played a significant role in speciation of crops, occurring frequently in nature, usually due to stress response.
In the 100 years since scientists discovered polyploidy, there has been rapid development of polyploid breeds. It is estimated that up to 80% of all flowering plants have polyploid varieties. 2 Common polyploid cultivars includes wheat, coffee, banana, strawberry, potato, etc.
Polyploidy has been researched since the early 1900’s. Scientists first used heat and electrical stress to induce those mechanisms. Today polyploidy is more commonly, and consistently, induced with radiation and stressing chemicals. Interestingly, induced polyploidy is explicitly exempt by most organic certification bodies. These types of breeds typically do not fall under ‘genetically modified’ until foreign, non-similar species, DNA is introduced to the plant cell.
These polyploids are called autopolyploid (same species), and plants made with dissimilar species are called allopolyploids. Cannadabis will also be exploring organic permitted cell fusion; this would allow breeding with two male plants, or two female plants.
In the past, the following horticulture benefits have been derived from polyploidy and cell fusion, which Cannadabis hopes to similarly apply to the cannabis plant: 3
- Increased yield, potency, flavour and vigour;
- 40% increase in trichome density: 70-100% increase in leaf terpenes. 4 Drought resistant strains;
- Ideal for outdoor growing and cultivators trying to limit water use.
Pest resistant strains;
- Pest resistance can occur through increased terpene production or through mechanistic resistance;
- Popular cultivars like the common banana were almost extinct from fusarium infections.
- Polyploidy and hybridisation were able to create a resistance strain of banana.
- Unique phenotyping;
- Cannadabis is hoping to unlock the cannabinoid profile of organic cannabis through polyploidy. One day this might include strains that produce 20% THCV, CBG, CBN etc.
Sterile hybrids; and
- Triploids produce infertile sex cells. This is the mechanism behind seedless watermelon. 5
The same can apply to cannabis. Strains can be developed that would never seed regardless of direct pollination; massive utility available to outdoor or indoor cultivators with seeding problems.
Cannadabis hopes to release their first polyploid strains in late 2020.
Premade and blended mediums
Cannadabis has begun manufacturing premade tissue culture mediums and are currently distributing them to Western Canadian horticulture stores and Amazon Marketplace; the mediums are a standard blend that works on 95%+ of the founders’ cultivars. The founders’ tissue culture experience is being provided to the public in both consumer and commercial grade products.
The introductory products show unfamiliar users how to do tissue culture at home, using proven methods that do not require expensive laboratory equipment. Besides what comes in the starter kit, the everyday home grower will usually have all the remaining materials at home. Commercial format mediums are intended for growers that want the best value and space savings.
Cultivators of any background can find information or help on tissue culture through the Cannadabis homepage. They are posting helpful videos and literature on cannabis tissue culture and hope to share the benefits with every grower. All horticulturalists, cannabis or not, can benefit from having their cloning area be 100x more efficient, through stackable containers. Furthermore, their mother plants can easily be maintained with minimal care. 100-1000 mother cultures can be stored within a refrigerator for 4-8 months, no adding nutrient or water. For larger cultivators, Cannadabis provides PGR matrices to more easily troubleshoot difficult cultivars. They also will custom blend and sterilise mediums to customer preference.
Cannadabis has begun developing an automated cell culture process for mass propagation of cultivars. The economies of scale of which are expected to change the supply chain of the entire cannabis industry. Automated cell culturing will provide starting materials to the industry at a fraction of the cost of inhouse cloning. Clones produced through cell culturing will also have the benefit of being totally sterile and free from disease.
Cannadabis has been offered an NRC-IRAP grant for initial developments of the process and are in early negotiations with a Canadian cannabis company to commercialise. The founders are expecting to file patents, mid 2020, and begin construction of a commercial scale process by mid-2021. Cannadabis anticipates that a 5000 sq ft facility will produce 5+ million clones annually, with minimal labour.
The project is looking to possibly incorporate the production of artificial seeds, which would simplify transportation and ease of storage for cultivators. They will also be developing cryogenic preservation methods. Cultivators around the world are encouraged to reach out to Cannadabis if they are looking to simplify their process, access cell culture benefits, and maximise growing space.
Working with Cannadabis’ cultured clones will be the most affordable, safe, and efficient way of acquiring starting material. Their services would include meristem culturing to remove systemic disease, and long-term storage of genetic inventory. Partners who end up with a pest could rest easy knowing their mother cultures will be perfectly preserved in tissue culture, and fifty thousand clones for the next crop are still on the way.
Delta 9 tissue culture pod
Cannadabis Medical and Delta 9 Cannabis have teamed up to provide an affordable, turnkey, tissue culture laboratory, complete with operating procedures, equipment, and cannabis medium recipes.
The two companies have co developed this system for their own commercial use and have recently made the system available for other cultivators. Both companies have recognised that the cannabis industry is still reliant on black market methods of propagation, and as a result, there have been countless incidents of crop and genetic loss in the legal industry; many of the stories circulating are understandably refuted by the companies experiencing such loss.
Rather than ignore the inevitable pest problems, the two companies are going toe to toe with mother nature, developing half century old technology and making it specifically for cannabis. Hopefully delivering the same modicum of control to the rest of the industry; cultivators slow to develop tissue culture science may soon find their genetics and crop totally destroyed by a single, often microscopic pest. On a commercial scale, these pests become essentially impossible to remove without the use of tissue culture.
With feet rooted in genuine care, Cannadabis and Delta 9 are prepared and excited to deliver a tissue culturing system to the global cannabis industry. They recognise the value and utility available to growers, and they also recognise that learning tissue culturing can feel out of reach for cultivators with no prior knowledge, or excess funding to hire an inhouse specialist.
Instead of missing out or paying specialists, cultivators can rely on Cannadabis and Delta 9 to deliver a ready to use laboratory, the development of which was based on maximising value for the growers.
The laboratory comes with only bare essentials and extensive, yet simple, operating procedures. Training materials will detail cannabis specific mediums, sanitation protocols, along with troubleshooting methods for finicky cultivars; an inexperienced grower will be comfortably blending and using mediums on the same day of commissioning. The whole system, equipment and all, will be much more affordable than hiring a tissue culture specialist.
Over the next three years, Cannadabis will be working to establish an expanded cultivation with the hope of supplying medical, organic, indoor grown cannabis to domestic and international markets.
They will also pioneer an original cell culture process that expects to be the most affordable source for starting materials in the world; Cannadabis is especially excited to deliver their polyploid cultivars as starting materials to industry members.
Cannadabis would like to offer an open invitation to all scientists, entrepreneurs, and industry professionals for collaboration. We are actively seeking partners who share a similar vision for the cannabis industry. Any professionals who are driven by a sense of genuine care and have a passion for cannabis medicine are encouraged to reach out.
CANNADABIS Medical INC
+1 306 552 4242
This article will appear in the first issue of Medical Cannabis Network which will be out in January. Click here to subscribe.
Cannadabis Medical INC is dedicated to the organic and holistic production of cannabis medicine and related technology.