micropropagation cannabis

Back to the roots: protocol for the photoautotrophic micropropagation of medicinal Cannabis


The aim of this protocol was to develop an alternative in vitro propagation system for Cannabis sativa L. by mimicking nursery-based vegetative propagation. Photoautotrophic micropropagation (PAM) was achieved on rockwool blocks as substrate combined with commercially available fertilizer suitable for cannabis cultivation. Stock plants were initiated after sterilisation in forced-ventilated glass jars which then provided a continuous supply of shoot tip and nodal cuttings. A 97.5% rooting rate of in vitro shoot tip cuttings and successful acclimatisation were achieved within 3 weeks in glass vessels with passive ventilation.

Key message

Cannabis is a difficult plant to grow in vitro, in particular habit is strongly influenced by culture conditions. A photo-autotropic micropropagation system was developed to overcome culture-induced problems.


Cannabis sativa L., rightfully also labelled as ‘the plant of the thousand and one molecules’ (Andre et al. 2016) has had a long tradition as a source of fibre, food and as a recreational and medicinal drug in the history of mankind. As more clinical studies prove its pharmaceutical value, governments are legalising medicinal cannabis and as a consequence demand is rising steadily (INCB, 2018).

Micropropagation as an aseptic technique has a high potential in cannabis propagation. It allows the rapid propagation of uniform materials of elite chemotypes free of diseases and pests. Valuable mother stock plants can be safe-guarded in culture and they enable shipment and transfer of high-quality germplasm. Although several micropropagation protocols have been published (Casano and Grassi 2009; Wang et al. 2009; Lata et al. 2016a, b), in vitro propagation of cannabis is challenging (Lata et al. 2017) and can result in off-types whose morphology does not resemble normal plantlets. The aim of this study was to find a reliable and productive micropropagation system that produces vigorous plants of high quality.


Initiation of aseptic stock cultures

Raise disease and pest-free cannabis stock plants in a glass house between 18 and 30 °C under a minimum of 18 h of light.

Take shoot tip cuttings below the third node (80–100 mm in size), remove the lowest leaf and rinse in tap water.

Surface sterilise in 0.5% NaOCl for 20 min in the laminar hood.

Rinse the plant material with sterile water and wash another three times for 10 min.

Prepare explants consisting of a shoot tip and two nodes (30–40 mm in size). Trim back leaf blades by two-thirds.

Transfer to either sterile forced ventilated glass jars or RITA containers (Vitropic, CIRAD, France).

Place the cultures in the growth chamber at 25 ± 1 °C with a 16 h photoperiod. Light intensity (PPFD—photosynthetic photon flux density) inside the vessels was around 70 µmol m −2 s −1 .

Maintenance of stock plants in forced ventilated glass jars

Place one quarter of a rockwool block (Grodan, The Netherlands, size 100 × 100 × 650 mm) into a glass preservation jar (1.5 or 2 L, 220 or 240 mm height) with a hinged lock and a rubber seal (Le Parfait super jars, France). The lid of the jar was modified so it could be forced-ventilated through sterile filters (Fig. 1a).

a Forced ventilated glass jars, b RITA system after 3 weeks culture, c regrowth of pruned shoots, d rooted shoots at time of transfer to glass house

Wet block with 20 mL of distilled water.

Autoclave the jars at 121 °C for 15 min.

After autoclaving, wet the block with 160 mL of sterile distilled water and plant three shoot tip cuttings into each block under sterile conditions.

Connect the closed jars via tubing to an air pump at a pressure of 1 bar. The air flow needs to pass by a bottle with distilled water to provide moisturised air. (Figure 1a) let the pump operate for 14 h per day, starting 2 h after the onset of the light period.

After 2 weeks, add 20 mL of nutrient solution (Canna Aqua Vega Fertilizer A + B Set, The Netherlands) to each jar; the nutrient solution is prepared with distilled water according to the manufacture’s instruction and the pH is set to 6.0–6.2 with NaOH before autoclaving at 121 °C for 15 min. The EC (electrical conductivity) of the nutrient solution is around 1.2.

After 6 to 7 weeks, tip-prune the plantlets for the first time and remove any wilted leaves.

Repeatedly prune plantlets as they grow bigger and use cuttings for rooting or setting up more stock plants.

Add water or nutrient solution (alternating) when rockwool blocks start to try out.

Maintenance of stock plants using the RITA-system

Set up a RITA container without the basket and bell with three rockwool blocks each (size 36 × 36 x 36 mm).

Wet blocks with 15 mL of distilled water.

Autoclave the containers at 121 °C for 15 min.

After autoclaving add 75 mL of sterile nutrient solution (described above) and plant two shoot tip cuttings into each block (6 explants per container) under sterile conditions.

Connect the jars via tubing to an air pump at a pressure of 1 bar. The air flow needs to pass by a bottle with distilled water to provide moisturized air. (Figure 1b) let the pump operate for 14 h per day, starting 2 h after the onset of the light period.

After 3 weeks, tip-prune the plantlets for the first time and remove any wilted leaves.

Repeatedly prune plantlets as they grow bigger and use cuttings for rooting or setting up more stock plants.

Add water or nutrient solution (alternating) when rockwool blocks start to try out.

Rooting and acclimatisation

Prepare 250 mL glass vessels (Hipp, Gmunden, Austria, 120 mm height) with vented lids (Magenta B-cap with 10 mm opening covered with adhesive microfiltration discs from TQPL, Hampshire, UK) with two rockwool blocks (size 20 x 20 x 40 mm).

Place the blocks upside down for stability and wet blocks with 10 mL of distilled water.

Autoclave vessels at 121 °C for 15 min.

After autoclaving, add 20 mL nutrient solution (described above).

Harvest shoot tips with three to four nodes from the in vitro stock plants and insert them into rockwool. Trim back large leaves. Use one shoot tip per block.

Place the cultures back in the growth chamber.

After 2 weeks add 10 mL of water to each jar.

Place lids upside down for the next 3 days to initiate the acclimatisation process.

Remove the lids completely and move the jars to a shelf with low lighting for the next 2 days to avoid drying out.

Pot up plantlets with the block into a general peat based potting mix into 100 mm pots (360 mL) and place them in a glass house with hand watering and a photoperiod of 16 h.

Results and discussion

After 5 weeks culture in forced-ventilated glass jars, 95% of the newly initiated shoot tips had grown on and were rooted and well developed. Cuttings from the in vitro stock plants were best taken once three or more nodes had regrown so that the lowest node(s) with axillary buds could remain for re-sprouting and to manage plant height. The stock cultures could be maintained this way for at least 6 months. The self-built preservation jars were more suited for the culture of cannabis as they provided more head space, on the other hand the RITA system was more practicable in terms of handling because of the wide opening. Shoot tip cuttings were the preferred source over nodal cuttings for the same reasons as in standard nursery propagation (Table 1). The biggest improvement through this PAM system was on plant quality.


A photoautotrophic in vitro propagation system has been developed that simply relies on industry-based fertilizer, rockwool blocks as substrate and forced ventilation.

Stock plants were initiated only once and provided a steady supply of shoot tips for propagation for at least 6 months.

In vitro plantlets were of excellent quality: leaves were dark green and compound, shoots had clearly defined internodes and there were no signs of hyperhydricity. Plantlets resembled their nursery analogues (Fig. 1c).

97.5% of in vitro shoot tip cuttings were rooted and acclimatized within 3 weeks inside the growth chamber.

Survival of rooted cuttings in the glass house was 100%.

The whole process did not require any sugars or vitamins. Not having to include sugars has two big advantages, (1) cultures are less prone to microbial contamination and (2) the acclimatisation step can be significantly reduced (Nguyen et al. 2016).

The whole process did not require any plant growth regulators either which almost certainly preserves genetic stability and overcomes fears of users that spontaneous mutations or somaclonal variation occur which are more likely associated with the use of growth regulators (Pierik 1997).


Andre CM, Hausman J-F, Guerriero G (2016) Cannabis sativa: the plant of the thousand and one molecules. Front Plant Sci 7:19.

Casano S, Grassi G (2009) Valutazione di terreni di coltura per la propagazione in vitro della canapa (Cannabis sativa). Italus Hortus 16:109–112

Lata H, Chandra S, Khan IA, ElSohly MA (2016a) In vitro propagation of Cannabis sativa L. and evaluation of regenerated plants for genetic fidelity and cannabinoids content for quality assurance. Methods in molecular biology. Springer, Clifton, pp 275–288

Lata H, Chandra S, Techen N, Khan IA, ElSohly MA (2016b) In vitro mass propagation of Cannabis sativa L.: a protocol refinement using novel aromatic cytokinin meta-topolin and the assessment of eco-physiological, biochemical and genetic fidelity of micropropagated plants. J Appl Res Med Aromat Plants.

Lata H, Chandra S, Khan IA, ElSohly MA (2017) Micropropagation of Cannabis sativa L.—an update. Cannabis sativa L.—botany and biotechnology. Springer International Publishing, Cham, pp 285–297

Nguyen QT, Xiao Y, Kozai T (2016) Photoautotrophic micropropagation. Plant factory. Elsevier, Netherlands, pp 271–283

Pierik RLM (1997) Somaclonal variation. In vitro culture of higher plants. Springer, Netherlands, pp 231–238

Wang R, He LS, Xia B, Tong JF, Li N, Peng F (2009) A micropropagation system for cloning of hemp (Cannabis Sativa L.) by shoot tip culture. Pak J Bot.


Open access funding provided by Austrian Science Fund (FWF). Thanks to C. Wawrosch for letting us use the self-built preservation jar system, to T. Hatfaludi, W. Kubelka and F. Malfent for fruitful discussions, to R. Hood for introducing us to this exciting field of research and to B. Forster for English editing. A. Kodym holds an Elise Richter Fellowship by the Austrian Science Fund (FWF) (V351-B22).

Author information


Department of Pharmacognosy, University of Vienna, 1090, Vienna, Austria

Andrea Kodym & Christian J. Leeb

Core Facility Botanical Garden, University of Vienna, 1030, Vienna, Austria

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Both authors contributed to the design and implementation of the work. AK has written the article, CL reviewed the final manuscript.

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The aim of this protocol was to develop an alternative in vitro propagation system for Cannabis sativa L. by mimicking nursery-based vegetative propagation

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.

Medical focus

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.

Automated propagation

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.

Future goals

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.

Alexander Calkins
+1 306 552 4242
[email protected]
Tweet @cannadabis

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Cannadabis Medical INC is dedicated to the organic and holistic production of cannabis medicine and related technology.