Cannabis tissue culture SOP
HomeStandard operating procedureCannabis tissue culture SOP
Standard operating procedure · forms included

Cannabis tissue culture SOP

Work through these jobs in order and you will be able to set up a clean tissue culture line, multiply a cultivar disease-free, and hand a tested plantlet to your grow room. Each job has a diagram and a form. A number in brackets is a source—you do not need to open it to do the step.

12 jobs14 formsHome + licensedPrint and follow
In this guide
01 Purpose and scope02 Evidence and limitations03 Preparing the workspace04 Selecting and setting up a laminar-flow hood05 Opening procedure06 Mix medium, autoclave, hold 7 days07 Explant preparation and surface sterilisation08 Plating, labelling and incubation09 Culture inspection and disposal10 Shoot multiplication and subculture11 Meristem dissection after process validation12 Rooting and acclimatisation13 Testing, intake and lot registration14 Weekly operating schedule15 Troubleshooting16 Source notes17 References
00

Purpose and scope

This is the working SOP. The tissue culture playbook is the longer guide. Follow this document if you are doing the work today.

Two setups share the same jobs. Home: one room, a still-air box or a cheap hood, a pressure canner. Licensed: the same jobs plus lot numbers and a disease test.

A small [n] after a number or a rule is a source. You do not need to open it to do the step. The list is at the end.[1]

SOP map. Do these in order.0Room + hoodClean the room. Buy and set up the hood.1Open the dayWipe. Warm the hood 20 min. Gown.2Mix mediumWeigh. pH. Agar. Pour. Autoclave.3Hold 7 daysWatch jars. Bin any that cloud.4Cut + bleachTake tissue. Surface-sterilise.5PlateOne piece, one jar. Label. Lid.6ScoutDay 7 and day 21. Bin dirty.7MultiplyRecut clean shoots every 3–4 weeks.8MeristemMicroscope cut if you need cleanup.9RootIBA in gel, or dip into a plug.10HardenDome. Open vents. Then a pot.11TestSend tissue for HpLVd qPCR.
Figure 1. The 12 jobs. Do 0–5 before you try meristems.
Paper pack. Fill these. Do not skip the lot number.F-01Roomdaily cleanF-02AutoclaveloadF-03MediabatchF-04Initiation/ plateF-05SubcultureF-06MeristemcutF-07ContaminationcullF-08RootingF-09HardenF-10Index/ qPCRF-11MotherintakeF-12HoodcheckF-13Gown/ entryF-14Lotregister
Figure 2. Fourteen forms. Print them. Write in pen. One lot number ties F-03, F-04, F-05 and F-10 together.
  • Lot number format: YYYY-MM-DD-CULTIVAR-NN. Example: 2026-08-14-MD-01.
  • One cultivar per hood session.
  • If a jar is doubtful, it is dirty. Bin it. Log it on F-07.
  • Do not call a plant clean unless F-10 has a negative qPCR result.
First run

Expect to lose most of the first batch. Published start-up losses sit between 45% and 95%.[4] Write what you did. Change one thing next time.

ExplantThe piece of plant you put in the jar.
MediumThe sterile jelly: salts, sugar, agar, optional hormone.
LotOne mix of medium, or one plating session, under one number.
Evidence assessment

Evidence and limitations

How sure is this paper?

We've gone to great lengths to keep these guides honest. One of the main ways we do that is self-review: we actively look for claims that are subjective, only lightly backed by literature, or based on grower practice rather than a controlled study — and we call those out instead of dressing them up as settled science.

Often there simply is no paper for the decision you're making. In those cases we're drawing on what other growers report and what has worked in our own rooms. That can still be useful — but it is not a lab proof. Do what works for your plants, your room, and your meters. If a table disagrees with your crop, believe the crop and log the difference.

Solid
Well supported by plant science, standards, or broad multi-source consensus
  • Core definitions and measurement units used in the paper
  • Safety-critical limits where occupational or standards sources are cited
Operational
What many growers and rooms actually run — start here, then tune
  • Numeric stage targets (light, climate, feed) as starting bands, not laws
  • SOPs that work in many rooms but need your genetics and meters
Grain of salt
Subjective, thin literature, single studies, or “this works for us” practice
  • Any single-number 'guaranteed' yield or potency claim without a multi-site trial
  • Controller setpoints copied from another facility without re-calibration

See something glaringly wrong? Tell us and we'll fix it. Please open a GitHub issue with the paper name and what looks off (include a source if you have one): Report an accuracy issue. Local law, labels, and licences always override any recipe here. Inline notes labelled grain of salt flag the highest-risk over-trust points in the text.

Job 0A

Preparing the workspace

Pick one small room or a closed corner. No carpet. No house plants. No cardboard boxes stored in it. A spare bedroom, a laundry, or a sealed cupboard bay will do.

How to treat the room, once, before first use1EmptyTake everything out.No plants. No cardboard.2WashWarm water + detergent.Walls, floor, bench, door.3Bleach10% household bleach.Wet 10 min. Then rinse.4Alcohol70% ethanol or IPA.Wipe. Let it dry.5SealTape gaps. No carpet.Door sweep if you can.6HoldHood on 30 min.Then start work.
Figure 3. Empty. Wash. Bleach. Alcohol. Seal. Then turn the hood on and wait.
Empty room ready for tissue culture
Example. What the room should look like before you bring plants in: bare floor, empty walls, one bench, door shut. No cardboard. No house plants.Grok Imagine · illustration
  1. 1
    Empty it
    Remove plants, food, cardboard, fabric, pet beds. Wipe dust off the ceiling fittings.
  2. 2
    Wash
    Warm water and household detergent on walls, floor, bench, door, window sill. Rinse.
  3. 3
    Bleach
    Mix 1 part household bleach (about 4–8% sodium hypochlorite) with 9 parts water. Wet surfaces for 10 minutes. Rinse. Wear gloves. Open a window.
  4. 4
    Alcohol
    Wipe hard surfaces with 70% ethanol or isopropyl alcohol. Let it dry.
  5. 5
    Seal
    Tape obvious wall gaps. Add a door sweep if the door has a gap. Cover carpet with sealed vinyl if you cannot rip it up.
  6. 6
    Rules for the room after this
    No eating. No outdoor shoes. No cardboard storage. Door shut while you work. One person at a time at the hood.

Weekly room treatment, after you are running

  • Floor and door handle: detergent, then 70% alcohol.
  • Bench and hood steel: 70% alcohol at the start of every work day (Job 1).
  • Bins: empty after every session. Do not leave dirty jars in the room overnight.
  • If you have a UV lamp in the hood: run it only with the sash down and nobody in the room. Never look at it. Wipe the tube monthly. UV is extra. It is not a substitute for the HEPA fan.
F-01 — Room daily clean
DateNameFloorBenchDoorHood steelBin emptiedSign
Tick each box. If you skip a box, do not start cultures that day.
Job 0B

Selecting and setting up a laminar-flow hood

A still-air box works. A horizontal laminar-flow hood is easier. You can buy one from China for a few hundred dollars if you order the right spec and check it on arrival.

What to order. Horizontal flow. H13 or H14 HEPA.Must haveHorizontal laminar flow (air toward you)H13 or H14 HEPA, plus a pre-filterMetal body. Not cardboard. Not a grow tentWork opening at least 400 mm wideAsk the sellerEN1822 or equivalent filter certificateFace velocity 0.30–0.50 m/s (photos + spec)Replacement HEPA size and priceVoltage 220–240 V if you are in AU/NZ/EUSkipVertical-only “biosafety” boxes for this jobNo pre-filter, foam-only, or “HEPA-like”UV as the only steriliser (it is extra, not the hood)Used filters. Buy a new sealed HEPA
Figure 4. Horizontal flow. H13 or H14 HEPA plus a pre-filter. Metal body. Certificate from the seller.
How the hood works. Air one way. You sit in front.pre+HEPAclean air sheet → 0.30–0.50 m/sYou sit here. Do not block the filter face. Do not put tall jars behind open work.you
Figure 5. Air is sucked through a pre-filter, then a HEPA, then blown toward you in one sheet. Work in that sheet. Do not block the filter.

What the words mean

  • Horizontal laminar flow. Air moves in one direction, from the filter face toward you. That is the type used for plant work. It protects the plant, not your lungs.
  • HEPA H13 / H14. A filter grade. H13 stops 99.95% of 0.3 µm particles. H14 is tighter. Either is fine. “HEPA-like” and foam pads are not.
  • Pre-filter. A cheap washable pad in front of the HEPA. It keeps dust off the expensive filter. If the listing has no pre-filter, skip it.
  • Face velocity 0.30–0.50 m/s. How fast the clean air comes out. Slower than 0.3 is weak. Faster than 0.6 dries tissue and can bounce dirt back.

Where to search

Search these exact phrases. Open the listing. Message the seller with the checklist below before you pay.[22]

Table 1. Search these. Listings change. Buy the spec, not the brand name.
SiteSearch phraseLink
AliExpresshorizontal laminar flow hood H14 HEPAaliexpress.com/w/wholesale-laminar-flow-hoods.html
AliExpress example classH13/H14 hoods around USD 260–500item 1005010452823851 (check current spec)
Alibabahorizontal laminar flow hood H14 plant tissue culturealibaba.com search
Alibaba branded classBIOBASE clean bench / LAFBIOBASE LAF listing
Prices move. Freight and a 220–240 V plug matter more than a $40 difference.

Message the seller this, copy-paste

Send this as one message. If they cannot answer, do not buy.

  • Is the cabinet horizontal laminar flow (not a biosafety cabinet)?
  • What HEPA grade? H13 or H14? Send the EN1822 (or equivalent) certificate for this filter lot.
  • Is there a separate pre-filter? What is the replacement HEPA size and price?
  • What face velocity at the work opening, in m/s?
  • Voltage and plug: I need 220–240 V (change this if you are on 110 V).
  • Work opening width in mm? I need at least 400 mm (16 in).
  • Photos of the filter gasket and the fan nameplate before shipping.

What to pay

  • Desktop / mini metal hood, H13/H14, 400–700 mm (16–28 in) wide: often USD 260–700 plus freight.
  • Full clean bench (BIOBASE class): often USD 800–2,000 plus crate freight.
  • A used Athena-style portable hood from the second-hand market can cost more than a new Chinese bench. Compare the filter spec, not the logo.
Do not buy

Cardboard mushroom boxes with a furnace filter. Vertical “nail salon” tables with no HEPA grade. Anything that says HEPA but shows a foam sheet. A biosafety cabinet unless you already know you need operator protection. It is the wrong tool and costs more.

When it arrives

Hood setup, first day1UnboxCheck the HEPA is sealed.No crushed corners.2PlaceLevel, solid bench.30 cm clear behind intake.3VoltageMatch the plate.Use a surge board.4First runOn for 30 minutes.Empty. No work yet.5Flow checkTissue strip in the stream.It should lean steadily out.6Wipe70% alcohol on steel.Never wet the HEPA face.7LogForm F-12.Date, velocity note, pass.8DailyOn 20 min before work.Wipe. Then start.
Figure 6. Unbox. Place. Match voltage. Run empty 30 minutes. Check the air stream with a tissue. Wipe steel only. Log F-12.
  1. 1
    Inspect
    HEPA frame not crushed. Plastic still on the filter face. No rattle in the fan.
  2. 2
    Place
    Level, solid bench. 30 cm (12 in) of free air behind or below the intake, depending on the model. Do not push it into a curtain.
  3. 3
    Power
    Read the plate. 220–240 V units die on 110 V. Use a surge-protected board.
  4. 4
    First run
    Empty hood. Fan on 30 minutes. Listen. Smell for burning.
  5. 5
    Flow check
    Hold a thin tissue strip in the work opening. It should lean steadily toward you (horizontal hood). If it flaps, flaps back, or hangs dead, message the seller before first use.
  6. 6
    Wipe
    70% alcohol on painted steel and the work tray. Never spray liquid into the HEPA face.
  7. 7
    Optional smoke
    A stick of incense 20 cm (8 in) in front of the filter. Smoke should leave in one sheet, no swirls back onto the bench.
  8. 8
    Log
    Fill F-12. If the tissue hangs dead, do not plate plants.
F-12 — Hood check
DateHours runTissue-strip pass?Noise / smellPre-filter cleaned?Sign
Do this on first setup, then monthly, and after any filter change.
No hood yet

Use a still-air box: a clear tub on its side, two arm holes, 70% alcohol wipe, fans off. Same jobs. Slower. Cheaper. The SOP steps do not change.

Job 1

Opening procedure

Do this every day you work, before any jar is opened.

Start of every work day1ClothesClean top. Hair tied.No outdoor shoes in the room.2Hood on20 minutes empty.Listen for the fan.3Wipe room70% alcohol, bench + door.Form F-01.4Wipe hoodSteel only. Back to front.Never the filter face.5GlovesNew nitrile. Spray 70%.Re-spray after any exit.6Tools inOnly today’s jars + tools.Waste bag on the left.
Figure 7. Clothes. Hood on 20 minutes. Wipe room. Wipe hood steel. New gloves. Only today’s tools.
  1. 1
    Clothes
    Clean long sleeves. Hair tied. No outdoor shoes in the room. Licensed lab: gown, hair cover, gloves. Log F-13.
  2. 2
    Hood on
    Empty. 20 minutes. If the fan sounds new or burnt, stop.
  3. 3
    F-01
    Floor, bench, door, hood steel, bin.
  4. 4
    Gloves
    New nitrile. Spray 70% alcohol. Spray again after you leave the room.
  5. 5
    Load the hood
    Today’s closed jars, tools, waste bag on the left. Nothing else.
F-13 — Gown / entry (licensed lab). Home: write “home” and skip unused columns.
DateTime inGownHair coverGlovesJewellery offTime outSign
Job 2

Mix medium, autoclave, hold 7 days

One litre. Full-strength MS for start and multiply. Half-strength MS if you are rooting in gel.

Mix and sterilise 1 litre of medium1Water800 mL RO or distilledin a 1 L flask or pot.2Salts + sugarMS 4.4 g. Sugar 30 g.Stir until clear.3ExtrasInositol 0.1 g.Charcoal 1 g optional.4pH5.6–5.8 now.Before agar.5Agar6–8 g (9.5 g if glassy).Heat to dissolve.6PourJars 1/3 full.Loose lids.7Autoclave121 °C / 15 psi / 20 min.Form F-02 + F-03.8Hold7 days on the shelf.Bin any that cloud.
Figure 8. Water. Salts. Sugar. pH. Agar. Pour. 121 °C (250 °F) / 103 kPa (15 psi) / 20 min. Hold 7 days.
Table 2. Default cannabis medium.
Ingredient1 litreNotes
RO or distilled waterstart 800 mL, top to 1 LNot tap
MS basal salts4.4 gHalf = 2.2 g for many rooting mixes
Sucrose30 gTable sugar is fine
myo-Inositol0.1 gSkip only if your MS already has it
Activated charcoal1 g optionalHelps with browning[1]
Agar6–8 g; 9.5 g if shoots go glassy[4]
PPM (optional)0.5–2 mLLabel rate. Not a meristem.[16]
pH5.6–5.8 before agarThen autoclave
Table 3. Hormones. Change one at a time.
If you wantAdd this after you know MS worksDose
Start (classic)TDZ + NAA1 µM + 0.5 µM[1][2]
Start (gentler)meta-Topolin0.48 mg/L[3][4]
Long multiplyNo hormone + extra calciumCa nitrate 0.71 g/L + Ca gluconate 1.35 g/L[4]
Root in gelIBA5 µM. More is worse.[1]
  1. 1
    Weigh
    Write every mass on F-03 before you pour.
  2. 2
    pH
    5.6–5.8 before agar. Dilute acid down. Dilute base up.
  3. 3
    Agar + heat
    Dissolve. Pour jars one-third full. Lids loose.
  4. 4
    Autoclave
    Stovetop canner that holds 103 kPa (15 psi), or an autoclave. 121 °C (250 °F), 103 kPa (15 psi), 20 minutes. Instant Pots do not count. Jars on a rack, not drowned.
  5. 5
    Cool
    Tighten lids when cool enough to handle. Label lot number on every jar.
  6. 6
    Hold
    Shelf 7 days. Any cloud or fuzz: bin the whole lot. Do not plate into it.
F-02 — Autoclave / canner load
DateLoad ID121 °C?15 psi?20 min?Cool / dryFail?Sign
If any of 121 / 15 / 20 is no, discard the load.
F-03 — Media batch
Lot no.DateMS gSugar gAgar gpHHormonePPM mLJars nDay-7 clear?Sign
Lot no. = YYYY-MM-DD-MED-NN. Carry this number onto F-04.
Job 3

Explant preparation and surface sterilisation

First runs: a stem piece with one bud, 10–15 mm (0.4–0.6 in). Not a meristem.

Surface-sterilise the plant piece1Cut10–15 mm tip or node.Strip big leaves.2Soap washTap + drop of soap.10–20 minutes.370% alcohol30–60 seconds.Then drain.4Bleach0.6–1% NaOCl + Tween.20–30 min, stir.5Rinse ×3Sterile water.3–5 min each.6Trim endsIn the hood.Cut off bleach-burn.
Figure 9. Cut. Soap. 70% alcohol 30–60 s. Bleach 20–30 min. Rinse three times. Trim the burned ends in the hood.
  1. 1
    Mother
    Vegetative. Scouted. Young if you can. One cultivar.
  2. 2
    Cut
    Morning. 10–15 mm (0.4–0.6 in). Strip large leaves. Keep wet.
  3. 3
    Soap wash
    Tap water + a drop of dish soap or Tween-20. 10–20 min.
  4. 4
    70% alcohol
    30–60 seconds. Drain.
  5. 5
    Bleach
    Holmes: 10% household bleach (about 0.625% NaOCl) + 0.1% Tween-20, 20 min, stir.[1] Das: 1% NaOCl for 30 min.[4] Do not go to 60 min.
  6. 6
    Rinse
    Sterile water, three times, 3–5 min each. Then into the hood.
  7. 7
    Trim
    Cut off white or cooked ends. That cut face goes into the gel.
Cannabis nodal explant
Example. A stem piece with one bud. This is the first-run cut.Grok Imagine · illustration
Job 4

Plating, labelling and incubation

One piece, one jar, until you know your rate.

Plate one piece1Open one jarIn the air stream.Lid face down, to the side.2Stand the pieceCut base in the gel.Bud above the gel.3Lid onAt once.Do not talk over it.4LabelCultivar, date, type.Form F-04.5Shelf24–26 °C, 16 h light.Do not open to look.6Day 7Scout. Bin cloudy.Form F-07.
Figure 10. Open one jar in the air stream. Stand the piece. Lid on. Label. Shelf. Scout day 7.
Inside the still-air box / flow hood, the sterile work zonearm openingsSTERILE FIELDwork only hereFlame / beadsterilize tools 5-10 s70% alcoholwipe surfaces + glovesScalpel + forcepsin alcohol jarSterile media jarssealed until useExplant dishsterile petri / tileDiscard piletrimmings + waste
Figure 11. Open jars only in the centre of the hood. Tools on the left. Waste on the left front.
  1. 1
    Air stream
    Work 10–20 cm (4–8 in) in front of the filter, not at the very edge.
  2. 2
    One lid
    Face down to the side. Never above the jar.
  3. 3
    Plant
    Cut base in the gel. Bud above.
  4. 4
    Lid
    On at once. Do not talk over the jar.
  5. 5
    Label
    Cultivar, date, explant type, lot number from F-03.
  6. 6
    Shelf
    24–26 °C (75–79 °F). 16–18 h light. About 70–100 µmol m⁻² s⁻¹. Do not open to look.
F-04 — Initiation / plate
Lot no.CultivarExplantn platedMedium lotDateDay-7 clean nDay-21 clean nSign
n plated must equal what you put in. Day-7 + dumped = n plated.
Job 5

Culture inspection and disposal

Day 7 and day 21. Look through the glass. Do not open a doubtful jar.

Scout. Three outcomes.KeepClear gel. Green tissue.No smell. No fuzz.Leave it closed.BinCloud, slime, or fuzz.Or a sour smell.Seal. Bag. Bin. Log F-07.WatchSlight haze, day 3–5.Do not open it.Check again day 7 and 21.
Figure 12. Keep. Bin. Watch. If you hesitate, it is a bin.
Dirty jars
Example. Left: bacteria. Right: fungus. Seal. Bag. Bin. Not in the hood you still use.Grok Imagine · illustration
Browning
Example. Brown ring = leak from the cut, not always microbes. Still do not open it next to clean work. Move survivors to fresh charcoal medium.Grok Imagine · illustration
Glassy shoots
Example. Wet, see-through leaves = too much hormone or water. Next multiply: no cytokinin, 9.5 g/L agar, vented lid.Grok Imagine · illustration
F-07 — Contamination cull
DateLot no.Jar IDBacteria / fungus / brown / glassAction (bin / watch)Sign
Every dumped jar gets a line. This is how you see if technique is improving.
Job 6

Shoot multiplication and subculture

Only from jars that were clean at day 21. New jar every time.

Every subculture adds mutations, reset before clones driftSAFE ≤ 5 cyclesDRIFT RISKmutations →12345678910subculture number
Figure 13. Recut. Fresh medium. New lot line on F-05.
Clean multiply jar
Example. What you are copying: green divided leaves, clear amber gel, no cloud.Grok Imagine · illustration
  1. 1
    Source
    A day-21 clean jar. One cultivar.
  2. 2
    Cut
    15–25 mm (0.6–1.0 in) shoot tip or a node with a visible bud.
  3. 3
    New jar
    Same medium or the no-hormone + calcium mix.[4]
  4. 4
    Density
    Home: 1–3 per jar. Licensed: what your F-07 rate allows.
  5. 5
    Clock
    3–4 weeks. Restart the line from a tested mother by about five recuts.[9]
F-05 — Subculture
DateFrom lotNew lotCultivarn movedMedium lotDay-21 clean nSign
Job 7

Meristem dissection after process validation

Microscope. 0.2–0.4 mm (0.008–0.016 in). Then a lab test. This job does not make a plant “clean” by itself.

Bench layout inside the hood or still-air boxAir blows from the back of the hood toward you. Do not put tall objects behind an open jar.BACK OF HOOD · filtered air comes from here1Tools restBead steriliser here.Left of the dish.2Microscope + dishStereo microscope, 10–40×.Black dish under the lens.Only open field on the bench.3Fresh mediumClosed jars. Open one onlywhen the piece is ready.4WastePeeled leaves, used blades.Do not reach over zone 2.5YouSit facing the hood.Hands enter from the front.Hands never pass over an open jar or the open dish.Home: same five zones inside the still-air box. Fans and AC off.
Figure 14. Same five zones as the hood layout. Microscope in the middle.
Tools. Use these, not kitchen knives.AStereo microscope10–40×. You cannotsee 0.3 mm without it.BFine forcepsLeft hand. Holds thestem 3–5 mm down.C#11 scalpelPointed blade. Righthand. Peels and cuts.DBlack dishDark so the paledome shows up.EBead steriliser~250 °C, 20 seconds.Safer than open flame.F70% alcoholGloves and bench.Does not kill HpLVd.
Figure 15. Microscope, fine forceps, #11 scalpel, black dish, beads, 70% alcohol.
Hand positions. Do not switch them.Left hand — forcepsHold the stem 3–5 mm below the tip.Rest your wrist on the bench.Do not squeeze the dome.This hand does not cut.Right hand — #11 scalpelBlade almost flat. Slide under a leaf.Peel outward, away from the dome.Final cut is one downward nick.Resterilise after every few peels.
Figure 16. Left holds. Right cuts.
The cut, one movement at a time1Take a 10–15 mm tipVegetative shoot.Strip large leaves now.2Onto the black dishOne drop sterile water.Under the microscope.3Hold with forcepsLeft hand, 3–5 mm down.Tip pointing up.4Peel the outer leavesBlade under the leaf.Flick the leaf away.5Peel the next pairSame motion. Slow.Stop at pale tissue.6Leave two tiny leavesThey shield the domeduring the bleach step.7cut hereCut under the domeOne nick, 0.2–0.4 mm.Do not saw.8Move to the gelForceps or a needle.Stand it up. Lid on.
Figure 17. Eight movements. Do not skip to the nick.
What you should see through the microscopeA StartGreen. Many leaves.Too big. Keep peeling.B Outer leaves offFewer leaves. Tip shows.Keep peeling.C Two tiny leaves leftPale dome visible.Stop. Bleach this.D After the cut0.2–0.4 mm piece.This goes on the gel.
Figure 18. Stop peeling when you see a pale dome and two tiny leaves.
Meristem tools
Example. Tool kit.Grok Imagine · illustration
Peeling a tip
Example. Left forceps, right scalpel. Same grip when the piece is 1 mm.Grok Imagine · illustration
Meristem dome
Example. Dome plus two tiny leaves. Cut just below. That piece is 0.2–0.4 mm.Grok Imagine · illustration
  1. 1
    New flush
    10–15 mm (0.4–0.6 in) vegetative tip. Strip large leaves before the hood.
  2. 2
    Sterile tools
    Beads ~250 °C (~480 °F), 20 s, then cool.
  3. 3
    Dish
    One drop sterile water. 10–20× then 30–40×.
  4. 4
    Peel
    Outer leaves off. Stop at two tiny leaves.
  5. 5
    Bleach that tip
    Shorter bleach than a woody node. Rinse.
  6. 6
    One nick
    0.2–0.4 mm (0.008–0.016 in).[6] Onto Holmes-type start medium.[1]
  7. 7
    Wait
    4–8 weeks. Then F-10. Expect about 41% negative at six months, not 100%.
F-06 — Meristem cut
DateMother IDn tipsn platedMedium lotn alive wk 8F-10 resultSign
Job 8

Rooting and acclimatisation

A shoot with no roots is not a plant. Inside a culture jar the air is nearly 100% humid, so the plant never developed the ability to close its stomata or build a waxy leaf surface—it had no reason to conserve water. Move it straight to open air and it wilts within hours, like a person stepping out of a steam room into cold dry air: the problem is the speed of the transition, not the destination. Lower the humidity in stages instead. This is acclimatisation.

Rooted plantlet
Example. White roots in clear gel. No brown ring.Grok Imagine · illustration
Harden
Example. Plugs under a dome. Vents half at day 7. Lid off at day 14.Grok Imagine · illustration
Acclimatization: step the humidity down, never drop it in one go40%60%80%100%d0d3d6d9d12d15d18d21dome shutcrack vents wider dailydome off, ambient airRoots and a working wax cuticle take ~2-3 weeks to form. Too-fast drying = wilt + death.
Figure 19. Do not put a jar plant onto a dry bench.
  1. 1
    Pick
    2–4 cm (0.8–1.6 in) shoot. Not glassy. Not brown.
  2. 2
    Root
    Option A: 5 µM IBA in gel, 2–4 weeks.[1] Option B: dip the base in 15 mM IBA for 2–4 min, then a sterile plug.[10] Option C: rockwool, ordinary fertiliser, vented jar, no sugar.[7]
  3. 3
    Plug
    Rockwool or coco soaked in mild veg nutrient, pH about 5.8.
  4. 4
    Dome
    Mist the walls. 16 h light. Gentle. 24 °C (75 °F).
  5. 5
    Vents
    Day 7 half. Day 9 full. Day 14 lid off.
  6. 6
    Pot
    Treat as a new clone. No 12-hour days for several weeks.
F-08 — Rooting
DateFrom lotMethod (gel / dip / no-sugar)nn rooted wk 4Sign
F-09 — Harden
Date outLotPlug typenn alive day 14Sign
Job 9

Testing, intake and lot registration

If F-10 is blank, the plant is a clone. Not a clean mother.

HpLVd clearance is brutally genotype-dependentOne meristem + thermotherapy protocol, 13 cultivars. Disease fully eradicated in just 5 of 13.0%25%50%75%100%100%Valarie94%Athena*69%FRB1.462%AnnaLee50%Wife26%Hybrid 914%Hybrid 514%EarlyPearly*“Athena” here is a cannabis STRAIN, not the Athena Ag kit. Source: 13-cultivar thermotherapy study, 2024-25.
Figure 20. Cut. Grow new leaves. Sample. Freeze a retain. Test again.
  1. 1
    Sample
    New fully expanded leaf or the leaf stalk, after the plant has grown.
  2. 2
    Lab
    A lab that runs cannabis Hop latent viroid RT-qPCR. Roots are the most reliable tissue if they will take them.
  3. 3
    Retest
    3–4 weeks later, and before a mother enters production.
  4. 4
    Retain
    Freeze spare tissue from every pass lot.
F-10 — Index / qPCR
DatePlant / lotTissueLabHpLVdOtherRetain IDPass?Sign
Pass = not detected. Anything else = quarantine or destroy. Do not write “clean” in this column.
F-11 — Mother intake / quarantine
Date inCultivarSourceRoomPests?F-10 dateF-10 resultRelease / destroySign
F-14 — Lot register
Lot no.OpenedClosedCultivarF-03F-04 nF-07 dumpedF-10Fate
One line per lot. Fate = multiply / root / mother / destroy.
Tools after a dirty plant

70% alcohol does not destroy Hop latent viroid RNA. Use 5–10% household bleach for 1–2 minutes, or 1000 ppm hypochlorous acid for 1 minute, then rinse.[6] Dedicated blades per cultivar if you can.

Job 10

Weekly operating schedule

Table 4. If the form is blank, the job did not happen.
DayHomeLicensed
MonF-01. Pour Sunday’s medium.F-01 + F-13. Media kitchen: F-02, F-03.
TuePlate 10–20 nodes. F-04.Plate from F-11 released mothers only.
WedMultiply day-21 cleans. F-05.One cultivar per hood. F-05.
ThuScout. F-07.Scout + photo lots. F-07.
FriRoot or dip. F-08. Send F-10 if due.qPCR batch. Quarantine fails.
Sat–SunDo not open jars.Alarms only.
  • Plan about 80–100 transfers per hour at a hood once you are practised.[15]
  • Test production mothers every 3–6 weeks (F-10).
  • Restart a production line by about five recuts from a tested backup.[9]
Job 11

Troubleshooting

Table 5. One variable per run.
What you seeChange this only
>50% dirty by day 7Slower hands. Younger mother. Smaller tip.
>50% dirty after day 14Meristem (Job 7). Optional 4–5% PPM soak.[16] Change mother substrate.
Brown in 48 hShorter bleach. Charcoal. Trim ends.
Glassy leavesNo cytokinin. 9.5 g/L agar. Vented lid.
No roots week 55 µM IBA, or a 15 mM dip, not more hormone.
Dies under the domeLonger half-vent. Rockwool, not a bubbler.
Looks fine, duds in flowerF-10 was skipped.
12

Source notes

Stars in the text point here. You do not need this page to run a day.

Photographs are illustrations. Diagrams are the ones to follow for size and order.

Legal

Only grow cannabis where you are allowed to. This SOP is horticultural, not legal advice.

Related papers
TC playbook
Start-to-finish cannabis tissue culture
Tissue culture
Clean up genetics from a speck of tissue
Cloning
Cuttings that root every time
Mother plants
Stock plants, HpLVd control & succession

References

  1. Holmes JE et al. (2021). Variables affecting shoot growth and plantlet recovery in tissue cultures of drug-type Cannabis sativa L. Frontiers in Plant Science, 12:732344. https://pmc.ncbi.nlm.nih.gov/articles/PMC8491305/
  2. Lata H, Chandra S, Khan IA, ElSohly MA (2009). Thidiazuron-induced high-frequency direct shoot organogenesis of Cannabis sativa L. In Vitro Cell. Dev. Biol.-Plant 45:12–19. https://doi.org/10.1007/s11627-008-9167-5
  3. Lata H, Chandra S, Techen N, Khan IA, ElSohly MA (2016). In vitro mass propagation of Cannabis sativa L.: a protocol refinement using novel aromatic cytokinin meta-topolin. J. Appl. Res. Med. Aromat. Plants 3:18–26. https://doi.org/10.1016/j.jarmap.2015.12.001
  4. Das R, Kretzschmar T, Mieog JC (2024). Importance of media composition and explant type in Cannabis sativa tissue culture. Plants 13(18):2544. https://doi.org/10.3390/plants13182544
  5. Atallah OO et al. (2023). Hop latent viroid: a hidden threat to the cannabis industry. Viruses / PMC. https://pmc.ncbi.nlm.nih.gov/articles/PMC10053334/
  6. Transmission, spread, longevity and management of hop latent viroid in cannabis in North America (2025). PMC. https://pmc.ncbi.nlm.nih.gov/articles/PMC11902214/
  7. Kodym A, Leeb CJ (2019). Back to the roots: protocol for the photoautotrophic micropropagation of medicinal Cannabis. Plant Cell Tiss. Organ Cult. 138:399–402. https://doi.org/10.1007/s11240-019-01635-1
  8. Kurtz LE et al. (2022). Ex vitro rooting of Cannabis sativa microcuttings and their performance compared to retip and stem cuttings. HortScience, 57(12):1576. https://journals.ashs.org/hortsci/view/journals/hortsci/57/12/article-p1576.xml
  9. Torkamaneh D et al. (2024). Somatic mutation accumulation in micropropagated cannabis is proportional to the number of subcultures. Plants, 13(14):1910. https://pmc.ncbi.nlm.nih.gov/articles/PMC11279941/
  10. Ioannidis K, Tomprou I, Mitsis V (2022). An alternative in vitro propagation protocol of Cannabis sativa L. presenting efficient rooting, for commercial production. Plants 11(10):1333. https://doi.org/10.3390/plants11101333
  11. Monthony AS, Page SR, Hesami M, Jones AMP (2021). The past, present and future of Cannabis sativa tissue culture. Plants 10:185. https://doi.org/10.3390/plants10010185
  12. Page SRG, Monthony AS, Jones AMP (2021). DKW basal salts improve micropropagation and callogenesis compared with MS basal salts in multiple commercial cultivars of Cannabis sativa. Botany 99:269–279. https://doi.org/10.1139/cjb-2020-0179
  13. Cryopreservation of shoot tips of elite cultivars of Cannabis sativa L. by droplet vitrification (2019). Medical Cannabis and Cannabinoids (Karger). https://pmc.ncbi.nlm.nih.gov/articles/PMC8489323/
  14. A temporary immersion system to improve Cannabis sativa micropropagation (2022). Frontiers in Plant Science. https://www.frontiersin.org/articles/10.3389/fpls.2022.895971/full
  15. Plant Cell Technology (2023–2024). How to tissue culture cannabis; Achieving disease-free cannabis stocks; How to sterilize your explants; Build a home tissue culture lab; PPM™ directions for use. (industry/manufacturer or non-journal source) https://plantcelltechnology.com/
  16. Plant Cell Technology. Plant Preservative Mixture (PPM™) product directions: 0.05–0.2% standard; 4–5% soak for endogenous load. (industry/manufacturer or non-journal source) https://plantcelltechnology.com/products/plant-preservative-mixture-ppm
  17. Athena Ag, Culture Kit, ROOTS/SHOOTS media, and Plant/Media/Lab Prep procedure (manufacturer documentation). (industry/manufacturer or non-journal source) https://www.athenaag.com/culture-kit
  18. Murashige T, Skoog F (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  19. Driver JA, Kuniyuki AH (1984). In vitro propagation of Paradox walnut rootstock. HortScience 19:507–509. https://doi.org/10.21273/HORTSCI.19.4.507
  20. Lubell-Brand JD, Kurtz LE, Brand MH (2021). An in vitro–ex vitro micropropagation system for hemp. HortTechnology 31:199–207. https://doi.org/10.21273/HORTTECH04779-20
  21. Punja ZK, Collyer D, Scott C, Lung S, Holmes J, Sutton D (2019). Pathogens and molds affecting production and quality of Cannabis sativa L. Front. Plant Sci. 10:1120. https://pmc.ncbi.nlm.nih.gov/articles/PMC6811654/
  22. AliExpress / Alibaba laminar-flow listings. Confirm H13/H14 + pre-filter + 0.30–0.50 m/s before paying. (industry/manufacturer or non-journal source) https://www.aliexpress.com/w/wholesale-laminar-flow-hoods.html

Citations marked in-text as [n] map to this list. Primary literature and official guidance except where noted. Cannabis tissue culture is strongly genotype-dependent, verify dilutions, hormone doses and local regulations against the primary sources before relying on them.