Tissue culture is a way to grow many identical cannabis plants from a tiny piece of a mother plant. The piece grows in a sterile container on a nutrient gel. Growers use it for three things: removing viroids and viruses, multiplying one cultivar into thousands of uniform plants, and storing a cultivar without keeping a mother plant alive.
Part 1 explains how tissue culture differs from taking cuttings and which hormones control it. It then walks through the four working stages, with the published recipes and results. Part 2 covers clearing a viroid infection, setting up a small lab, problems specific to cannabis, storage, and running a culture room in Thai heat.
This article draws on eight published studies rather than on one source article. They are listed under References at the end. The numbers in square brackets point to them.
1. What tissue culture is and why cannabis growers use it
Most growers multiply a cultivar by traditional cloning. You cut a branch, dip it in rooting hormone and wait for roots. This has worked for decades. Its weakness is that each cutting carries every problem its mother has.
Those problems include viruses and viroids. Viroids are tiny infectious strands of RNA, even smaller than a virus. The most damaging one in cannabis is Hop Latent Viroid (HLVd). Cuttings also carry bacteria living inside the tissue.
Over years of taking cuttings from cuttings, small genetic changes also build up. This is called genetic drift, one of the causes of strain degradation. A cutting passes these changes on too.
Tissue culture, also called micropropagation, starts from a much smaller piece. Often this is the meristem tip, the growing point at the very end of a shoot. It can be as small as 0.4–0.5 mm. The piece you place into culture is called the explant.
The explant grows in a sterile container on a nutrient gel, under controlled light and temperature. From one explant you can raise large numbers of genetically identical plantlets, the small plants that form inside the container. If the explant is small enough, the plantlets are free of the pathogens that cuttings pass on.
The method is not new. Murashige and Skoog published their nutrient medium in 1962, first for tobacco tissue, and it is still the basic recipe today [5]. Bananas, orchids, potatoes and strawberries have been propagated this way commercially for decades. Cannabis growers adopted it more recently, for three reasons:
- HLVd spread unnoticed through commercial farms. It was first reported in cannabis in California (USA) in 2019 [3]. A 2023 review reports that about 90% of cannabis facilities in California tested positive. About 30% of plants in each facility showed symptoms. The review estimates industry losses of up to USD 4 billion a year [1].
- Medical supply chains require proof of origin. Farms working to GACP or GMP must trace every product back to its mother plant. GACP (Good Agricultural and Collection Practice) is the farm standard for medicinal plants. GMP (Good Manufacturing Practice) is the standard for making medicines.
- Large licensed farms need volume. One facility may need tens of thousands of uniform plants per cycle.
Translator’s note: Thailand already has a commercial tissue-culture industry for orchids and other ornamental plants. Whether any lab sells clean cannabis plantlets or tests cannabis for HLVd is a separate question. We could not find a Thai lab that publicly offers either service. Until you have found such a service, plan to do small-scale work yourself or to manage a mother plant very carefully.
What to do: Before you buy any equipment, ask in the forum’s Tissue Culture section whether any member has used a Thai lab for cannabis plantlets or HLVd testing. Ask what it cost and how long it took.
2. Cuttings and tissue culture side by side
| Traditional cutting | Tissue culture | |
|---|---|---|
| What you start from | 10–15 cm stem cutting | 0.4–5 mm meristem tip, or a stem segment with a bud |
| Diseases | Carries everything the mother carries | Can remove viroids, viruses and bacteria |
| Output per mother | 10–30 cuttings per session | Hundreds to thousands of plantlets from one explant |
| Time to a rooted plant | 10–14 days | 6–12 weeks, but many plants at once |
| Genetic stability | Drifts over years | Stable when new shoots grow from existing shoots |
| Space | Mother room | Small lab |
| Skill | Beginner | Intermediate to advanced |
| Cost | Minimal | Moderate to high |
| Long-term storage | Mother plant must stay alive | Cultures can be held for months |
Read the table as a choice between two tools. Tissue culture does not replace cuttings everywhere. It solves specific problems that cuttings cannot solve: removing viroids and viruses, producing very large uniform batches, and holding a cultivar without a living mother.
Translator’s note: In Thailand and Southeast Asia, many growers start a new cultivar by buying cuttings from another farm. This is the fastest way to bring in spider mites and thrips, powdery mildew or HLVd. You can also end up with a plant sold under the wrong strain name. Keep bought cuttings in a separate space, away from your mothers. Check them under magnification before they join the main room. Ask the seller whether the mother was tested for HLVd, by which lab and when.
What to do: Write down the problems you actually have, such as viroid symptoms, uneven batches or a mother room you cannot afford. If none of them applies, cuttings are probably still the right tool for you.
3. The biology behind micropropagation
Every living plant cell can rebuild a whole plant
Plant cells are totipotent. This means any living plant cell holds the full genetic information needed to regrow a complete plant. Animal cells lose this ability early in development. Totipotency is the reason tissue culture works at all.
On a gel with the right hormone balance, plant tissue can be pushed in four directions. It can multiply into callus, a lump of unorganized cells. It can form shoots, or it can form roots. In advanced work, it can form complete embryos from ordinary body cells instead of from seed (somatic embryos).
Two hormone groups control the culture
The direction a culture takes depends mainly on the ratio between two groups of plant hormones.
| Hormone group | Examples | Effect at high concentration |
|---|---|---|
| Cytokinins (hormones that drive shoot and bud formation) | BAP (6-benzylaminopurine), kinetin, TDZ (thidiazuron) | More new shoots |
| Auxins (hormones that drive root formation; IBA is also sold as rooting powder for cuttings) | IBA (indole-3-butyric acid), NAA (naphthaleneacetic acid), 2,4-D | More roots |
- High cytokinin, low auxin: many new shoots from one explant (multiplication).
- Low cytokinin, high auxin: the shoots grow roots (rooting).
- Balanced, or high auxin: callus forms.
This ratio is the most important setting you control. When it is right, shoots multiply efficiently. When it is wrong, you get callus or no growth at all. In cannabis, callus rarely turns back into shoots, and it carries a genetic risk (section 7, Part 2).
The recipes in section 4 follow this logic stage by stage:
| Stage | Cytokinin | Auxin | Balance |
|---|---|---|---|
| Initiation | 0.5 mg/L BAP | 0.1 mg/L NAA | 5 : 1, mild push toward shoots |
| Multiplication (BAP recipe) | 1.0–2.0 mg/L BAP | 0.1 mg/L NAA | 10–20 : 1, strong push toward shoots |
| Multiplication (TDZ recipe) | 0.5 µM TDZ | None | Cytokinin only |
| Rooting | None | 2.5 µM IBA | Auxin only |
Genotype decides a great deal
Genotype means the genetic makeup of a particular cultivar or plant. Most published cannabis recipes were fine-tuned on one cultivar. Other genotypes can respond very differently.
Monthony and colleagues at the University of Guelph (Canada) published a review of cannabis tissue culture in 2021 [4]. In the hemp studies they reviewed, callus formation on petioles (leaf stalks) ranged from 27% to 83%, depending on cultivar. Regeneration from hypocotyls ranged from 2% to 71%, depending on source tissue and cultivar. The hypocotyl is the seedling stem below the first leaves.
So a recipe that works very well on one cultivar may fail completely on another. Treat every published recipe in this article as a starting point. Then adjust it on your own genetics.
What to do: For each new cultivar, run the recipe on a small test batch first. Count how many explants respond before you scale up.
4. The four stages of cannabis micropropagation
Stage 0: Prepare the mother plant
Grow the mother under clean conditions for at least 2 weeks before you take tissue. Use no foliar sprays and no pesticides in that time. Feed a balanced, moderate-strength solution. Keep her in active vegetative growth under 18/6 light (18 hours on, 6 hours off). The aim is fresh, fast-growing shoots with no spray residue on them.
Where possible, test the mother for HLVd by RT-qPCR before you start. RT-qPCR is a lab test that detects and measures the viroid’s genetic material. The result tells you which explant to use. A mother that tested clean lets you use the easier stem segments. An infected or untested mother needs meristem tips (Stage 1).
Translator’s note: When you treat mothers outside this two-week window, use only pesticides registered with the Department of Agriculture (กรมวิชาการเกษตร). Use them exactly as the label says. See what may legally be sprayed under Thai GACP. If you cannot find an HLVd test in Thailand, treat the mother as possibly infected and work from meristem tips.
What to do: Mark the mother you will use, stop all sprays on her, and write down the date. Start Stage 1 no earlier than 14 days later.
Stage 1: Initiation — starting a clean culture
This is the most important stage, and the one that fails most often. The goal is to move a piece of plant tissue into a sterile container without any microbes coming with it.
Choosing the explant
- Shoot tips (meristems) are the choice for removing viroids. The dome at the very tip of a growing shoot is often free of pathogens, even when the rest of the plant is infected. The reason is that the plant’s internal transport channels (vascular tissue) have not yet grown into it.
- Punja and colleagues (Simon Fraser University, Canada) used tips of 0.4 mm with two or fewer leaf primordia [2]. Leaf primordia are the tiny bumps that later become leaves.
- A 2023 review by Adkar-Purushothama and colleagues in the journal Viruses compared ways of removing HLVd [1]. It found that culturing tips under 0.5 mm, combined with cold treatment, worked better than either method alone.
- You need a magnifier or a microscope to cut tissue this small.
- Nodal segments are 5–10 mm pieces of stem with a leaf joint and its bud. They are easier to handle, and beginners succeed with them more often. They do not remove viroids. Use them for multiplication when the mother has already tested clean.
Cleaning the surface of the explant (standard method)
- Rinse under running tap water for 5 minutes.
- Dip in 70% ethanol for 30 seconds.
- Soak for 10–15 minutes in 1–2% sodium hypochlorite (household bleach diluted about 1:5) with 1–2 drops of Tween-20. Tween-20 is a mild detergent that helps the bleach wet the whole surface.
- Rinse 3 times in sterile distilled water inside the hood or box, to wash off the bleach.
- Trim to final size with a sterile scalpel.
- Place on initiation medium.
Translator’s note: Household bleach sold in Thailand varies in strength. Read the sodium hypochlorite percentage on the label and dilute it to 1–2%. For example, a 6% product mixed 1 part bleach to 4 parts water gives 1.2%. Use plain bleach with no added scent or thickener.
A common initiation medium (based on MS)
- Murashige & Skoog (MS) basal salts and vitamins, the standard mineral and vitamin mix for plant tissue [5]
- 30 g/L sucrose (ordinary sugar, the tissue’s energy source inside the container)
- 0.5 mg/L BAP (cytokinin)
- 0.1 mg/L NAA (auxin)
- 7 g/L agar (a seaweed-based gel that sets the medium)
- pH adjusted to 5.7–5.8 before you sterilize the medium in an autoclave (a pressurized steam sterilizer) or pressure cooker
Hormones are measured from a stock solution of 1 mg/mL, stored frozen (method in section 6, Part 2). At that strength, 1 mL of stock in 1 litre of medium gives 1 mg/L. So this recipe needs 0.5 mL of BAP stock and 0.1 mL of NAA stock per litre.
What to expect. Within 2–4 weeks the explant should turn green and grow, with new leaves coming from the meristem or the node. Cloudy or yellow medium means the culture is contaminated. So does fuzzy growth. Discard contaminated containers without opening them in your work area, so spores do not spread.
Losses at this stage are normal and can be heavy. Expect them to be heavier in a still air box than in a laminar flow hood. A still air box is a closed, clear plastic box with arm holes. A laminar flow hood is a cabinet that blows filtered air across the work surface. Plan for losses: start 10–20 explants for every culture you need to keep. Early failures do not prove that the method does not work.
What to do: Label every container with the date, the mother and the explant type. At 4 weeks, count the clean and the contaminated containers. This gives you your own loss rate for planning.
Stage 2: Multiplication
Multiplication begins once Stage 1 has given you clean, growing cultures. Every 3–4 weeks, open each container in the hood or box. Divide each cluster into single shoots or small clusters, and move each piece to fresh medium. Each of these transfers is called a subculture.
Multiplication medium (BAP-based, general use)
- MS basal salts
- 30 g/L sucrose
- 1.0–2.0 mg/L BAP (1–2 mL of 1 mg/mL stock per litre)
- 0.1 mg/L NAA (0.1 mL of stock per litre)
- 7 g/L agar
- pH 5.7–5.8
The published cannabis recipe with TDZ. The most widely cited cannabis recipe comes from Lata and colleagues (2009) [6], a research team at the University of Mississippi (USA). They used MS medium with 0.5 µM thidiazuron (TDZ), about 0.11 mg/L. µM (micromolar) measures concentration by number of molecules rather than by weight.
At that dose, 100% of explants responded, producing an average of 12.6 shoots per explant.
Note how low that dose is. TDZ is very strong, and more does not work better. Too much TDZ directly causes glassy, water-soaked shoots (hyperhydricity, section 7, Part 2). Measuring 0.11 mL is difficult, so make a weaker TDZ stock: at 0.1 mg/mL you add 1.1 mL per litre.
Many cultures decline at this stage. Most introductions leave this out. Monthony and colleagues report a 74–82% drop in the number of regenerated explants taken from Stage 1 plants [4]. The decline shows up as glassy shoots, unwanted callus, and cultures that simply die.
Multiplication in cannabis is real, but it does not double neatly every cycle the way a spreadsheet might suggest. Base your plan on what your own cultivar actually does over several cycles, not on a theoretical rate.
What to do: Keep one record sheet per cultivar. At each subculture, write down shoots in and shoots out. Also record how many shoots turned glassy, formed callus or died.
Stage 3: Rooting
When you have enough shoots, move single shoots to a rooting medium. The hormone balance now switches from cytokinin to auxin.
Rooting medium (Lata and colleagues, 2009) [6]
- Half-strength MS salts
- 500 mg/L activated charcoal (a fine charcoal powder that soaks up browning compounds and some hormones)
- 2.5 µM IBA (about 0.5 mg/L, or 0.5 mL of 1 mg/mL stock per litre)
- No cytokinin
- 7 g/L agar
- pH 5.7–5.8
That recipe gave 95% rooting. Roots usually appear within 2–3 weeks. The plantlet is ready for Stage 4 when its roots are 1–2 cm long with visible root hairs.
Alternative: rooting outside the container (ex vitro). Some methods skip the rooting medium. Shoots come straight off the multiplication medium and are dipped in rooting hormone (gel or IBA powder). They then go into sterile substrate under a humidity dome. This saves time and medium cost. In return, you must control humidity and temperature more tightly during the change.
Translator’s note: Rooting outside the container is close to the cloning many Thai growers already do. In the hot season, a dome in direct sun or in a room without air conditioning can overheat quickly. Keep the dome in a cooled room and put a thermometer inside it.
What to do: Split one batch of a single cultivar between the two methods. After 3 weeks, compare how many shoots rooted and survived with each.
Stage 4: Acclimatization (hardening off)
Many growers lose plants at this stage. Inside the container, plantlets lived at near-100% humidity and a constant temperature. They were fed by sugar in the gel and never felt moving air. A sudden move into normal room air is too big a change, so they must get used to it in steps.
Acclimatization steps
- Take the plantlet out and gently wash the agar off the roots in lukewarm water. Sugary agar left on the roots feeds mould.
- Plant it in pre-moistened sterile substrate: peat and perlite at 70:30, or rockwool.
- Place it under a humidity dome at 90–95% relative humidity, 22–25 °C and 100–150 PPFD. PPFD measures how much usable light reaches the plant, in µmol/m²/s.
- Days 1–7: keep the dome closed.
- Days 7–14: start opening the dome slightly, for 1 hour, then 2 hours, then 4 hours.
- Days 14–21: remove the dome completely.
- From week 4: normal vegetative conditions and feeding.
With this general approach, Lata and colleagues reported that their rooted plants were successfully acclimatized in soil [6]. They reported 90% survival for plants regrown from encapsulated stem segments, which are pieces sealed in a gel bead (section 8, Part 2) [7]. These are lab results from a fine-tuned method. Losses at home are usually higher.
Most home losses come from damping off. This is a rot at the stem base of young plants, caused by fungi and water moulds in the substrate. The two controls that matter most are sterile substrate and careful, limited watering.
Translator’s note: Holding 22–25 °C under a dome is difficult in a 30–35 °C room without air conditioning, so do this stage in a cooled room. In the rainy season the air is already humid, and the substrate stays wet longer. Water less often than you would in the dry season.
What to do: For each batch, count the surviving plantlets at day 21 and at week 8. Write down the cause of every loss, so you know which step to fix.
If you have worked through any of these stages with cannabis, post your recipe, cultivar and counts below this topic. Questions are welcome too.
References
- Adkar-Purushothama, C.R., Sano, T., & Perreault, J.-P. (2023). Hop latent viroid: A hidden threat to the cannabis industry. Viruses, 15(3), 681.
- Punja, Z.K., Scott, C., Tso, H.H., Munz, J., & Buirs, L. (2025). Transmission, spread, longevity and management of hop latent viroid, a widespread and destructive pathogen affecting cannabis (Cannabis sativa L.) plants in North America. Plants, 14(5), 830.
- Bektaş, A., Hardwick, K.M., Waterman, K., & Kristof, J. (2019). Occurrence of hop latent viroid in Cannabis sativa with symptoms of cannabis stunting disease in California. Plant Disease, 103(10), 2699.
- Monthony, A.S., Page, S.R., Hesami, M., & Jones, A.M.P. (2021). The past, present and future of Cannabis sativa tissue culture. Plants, 10(1), 185.
- Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15(3), 473–497.
- Lata, H., Chandra, S., Khan, I.A., & ElSohly, M.A. (2009). Thidiazuron-induced high-frequency direct shoot organogenesis of Cannabis sativa L. In Vitro Cellular & Developmental Biology – Plant, 45(1), 12–19.
- Lata, H., Chandra, S., Mehmedic, Z., Khan, I.A., & ElSohly, M.A. (2012). In vitro germplasm conservation of high Δ9-tetrahydrocannabinol yielding elite clones of Cannabis sativa L. under slow growth conditions. Acta Physiologiae Plantarum, 34(2), 743–750. Client Challenge
- Lata, H., Chandra, S., Techen, N., Khan, I.A., & ElSohly, M.A. (2011). Molecular analysis of genetic fidelity in Cannabis sativa L. plants grown from synthetic (encapsulated) seeds following in vitro storage. Biotechnology Letters, 33(12), 2503–2508.
This article is part of the Asiannabis Community educational series. It is for legal, educational use only, in places where growing cannabis is permitted.
Continued in Part 2: Tissue Culture and Micropropagation for Cannabis: Clearing Hop Latent Viroid, a Home Lab, Storage and Tropical Conditions (Part 2 of 2)