Cannabis tissue culture SOPCannabis tissue culture SOP

Purpose and scope

This document is the SOP that you use to do the work. The tissue culture playbook is a longer document.

Two setups use the same tasks. A home setup has one room, a still-air box or a hood with a low price, and a pressure canner. A setup in a licensed facility has the same tasks. It also has lot numbers and a disease test.

A small ^[n] after a number or an instruction shows a source. It is not necessary to open the source to do the step. The list of sources is at the end.^[1]

• The format of the lot number is YYYY-MM-DD-CULTIVAR-NN. Example: 2026-08-14-MD-01.
• In each work period at the hood, use only one cultivar.
• If you are not sure that a jar is clean, the jar is dirty. Discard the jar. Record the jar on F-07.
• Do not think that a plant is clean if F-10 does not show a negative qPCR result.

Evidence and limitations

Prepare the work area

Select one small room or a closed corner. The area must not have a carpet or a houseplant. Do not keep cardboard boxes in the area. Each of these areas is satisfactory: a bedroom that you do not use, a laundry or a sealed space in a cupboard.

1. 1
   Make the room empty

   Remove the plants, the food, the cardboard, the fabric and the pet beds from the room. Remove the dust from the fittings of the ceiling.
2. 2
   Clean with detergent

   Use warm water and household detergent on the walls, the floor, the bench, the door and the window sill. Then flush the surfaces with water.
3. 3
   Clean with bleach

   Mix 1 part household bleach (approximately 4–8% sodium hypochlorite) with 9 parts water. Apply the solution to the surfaces and keep them wet for 10 minutes. Flush the surfaces with water. Put on gloves. Open a window.
4. 4
   Clean with alcohol

   Clean the hard surfaces with 70% ethanol or isopropyl alcohol. Let the surfaces dry.
5. 5
   Seal the gaps

   Use tape to seal the gaps that you can see in the walls. If the door has a gap, install a door sweep. If you cannot remove the carpet, put sealed vinyl on it.
6. 6
   Instructions for the room after this step

   Do not eat in the room. Do not go into the room in outdoor shoes. Do not keep cardboard in the room. Keep the door closed while you do the work. Only one person at a time can be at the hood.

Treatment of the room each week, after you start the work

• Clean the floor and the door handle with detergent. Then clean them with 70% alcohol.
• Clean the bench and the steel of the hood with 70% alcohol at the start of each work day (Task 1).
• Remove the waste from the bins after each work period. Do not let dirty jars stay in the room at night.
• The hood can have a UV lamp. Operate the UV lamp only when the sash is down and no person is in the room. Make sure that no UV light goes into your eyes. Clean the tube each month. The UV lamp gives more protection, but it does not replace the HEPA fan.

Selection and installation of a laminar-flow hood

You can use a still-air box. A horizontal laminar-flow hood is easier to use. You can purchase a hood from China at a low price. Make sure that you tell the seller the correct specification. When you receive the hood, do a check of it.

Definitions of the terms

• Horizontal laminar flow. The air moves in one direction, from the face of the filter to you. Use this type for work with plants. The hood gives protection to the plant. It does not give protection to your lungs.
• HEPA H13 / H14. A grade of filter. An H13 filter stops 99.95% of the particles of 0.3 µm. An H14 filter stops a larger percentage of the particles. Each grade is satisfactory. A “HEPA-like” filter and foam pads are not satisfactory.
• Pre-filter. A pad with a low price that you can clean. It is in front of the HEPA filter. It keeps dust away from the HEPA filter. The HEPA filter has a high price. If the listing has no pre-filter, do not select that listing.
• Face velocity 0.30–0.50 m/s. The speed of the clean air that comes out of the filter. A speed of less than 0.3 m/s is not sufficient. A speed of more than 0.6 m/s makes the tissue dry and can cause dirt to go back into the work area.

Where to find a hood

Use these terms to find hoods. Do not change the terms. Open the listing. Send the checklist below to the seller before you purchase the hood.^[22]

Message to the seller

Send the list below to the seller as one message. If the seller cannot give the information, do not purchase the hood.

• Give the type of the cabinet. It must be a horizontal laminar-flow cabinet and not a biosafety cabinet.
• Give the HEPA grade: H13 or H14. Send the EN1822 certificate (or an equivalent certificate) for the lot of this filter.
• The hood must have a pre-filter and a HEPA filter as two parts. Give the size and the price of a replacement HEPA filter.
• Give the face velocity at the work opening, in m/s.
• The voltage and the plug must be for 220–240 V (change this value if your power supply is 110 V).
• Give the width of the work opening in mm. The width must be a minimum of 400 mm (16 in).
• Send photos of the gasket of the filter and the nameplate of the fan before the shipping.

Prices

• A small metal hood for a bench has an H13 or H14 filter and a width of 400–700 mm (16–28 in). The price is frequently USD 260–700, plus freight.
• A full-size clean bench (BIOBASE type): the price is frequently USD 800–2,000, plus freight in a crate.
• A portable hood of the Athena type, from the market for used equipment, can have a higher price than a new bench from China. Compare the specifications of the filter, not the brand name.

When you receive the hood

1. 1
   Examine the hood

   Make sure that the frame of the HEPA filter has no damage. Make sure that the plastic stays on the face of the filter. Make sure that the fan does not make a rattle.
2. 2
   Put the hood in position

   Use a bench that is level and stable. Keep 30 cm (12 in) of free air behind the intake or below the intake. The correct position is different for different models. Do not push the hood against a curtain.
3. 3
   Power

   Read the nameplate. A unit for 220–240 V becomes defective when you connect it to 110 V. Use a power board with surge protection.
4. 4
   First operation

   Make sure that the hood is empty. Operate the fan for 30 minutes. Listen to the fan. Smell the air for a burned odor.
5. 5
   Check of the airflow

   Hold a thin strip of tissue paper in the work opening. The strip must bend in the direction of the airflow, to you, and stay stable (horizontal hood). The strip must not move up and down, go back to the hood or hang down with no movement. If the strip does this, send a message to the seller before you use the hood.
6. 6
   Clean the hood

   Clean the painted steel and the work tray with 70% alcohol. Do not spray liquid on the face of the HEPA filter.
7. 7
   Optional test with smoke

   Hold an incense stick 20 cm (8 in) in front of the filter. The smoke must move away from the filter in one sheet. It must not go back to the bench.
8. 8
   Record the data

   Write the data on F-12. If the tissue paper hangs down with no movement, do not put plants in jars.

Opening procedure

Do this task each day that you do the work. Do it before you open a jar.

1. 1
   Clothing

   Put on clean clothing with long sleeves. Make sure that your hair does not hang down. Do not go into the room in outdoor shoes. In a licensed lab, put on a gown, a hair cover and gloves. Write the data on F-13.
2. 2
   Start the hood

   Make sure that the hood is empty. Operate the hood for 20 minutes. If the fan makes a new noise or a burned odor, stop the hood.
3. 3
   Form F-01

   Clean the floor, the bench, the door, the steel of the hood and the bin. Record the work on the form.
4. 4
   Gloves

   Put on new nitrile gloves. Spray the gloves with 70% alcohol. Spray the gloves again after you go out of the room.
5. 5
   Put the items in the hood

   Put the closed jars, the tools and the waste bag for this work period on the left. Do not put other items in the hood.

Mix the medium, sterilize it and keep it for 7 days

The quantity in this task is one liter. Use full-strength MS to start cultures and for multiplication. Use half-strength MS to make roots in gel.

1. 1
   Weigh

   Write each mass on F-03 before you put the medium in the jars.
2. 2
   pH

   Set the pH to 5.6–5.8 before you add agar. To decrease the pH, add dilute acid. To increase the pH, add dilute base.
3. 3
   Agar and heat

   Dissolve the agar with heat. Fill each jar to one third with medium. Keep the lids loose.
4. 4
   Sterilize

   Use a stovetop pressure canner that holds 103 kPa (15 psi), or an autoclave. Sterilize at 121 °C (250 °F) and 103 kPa (15 psi) for 20 minutes. An Instant Pot is not satisfactory for this step. Put the jars on a rack. The jars must not be in the water.
5. 5
   Let the jars become cool

   When the jars are sufficiently cool to touch, tighten the lids. Put a label with the lot number on each jar.
6. 6
   Hold

   Keep the jars on a shelf for 7 days. If a jar has a cloud or mold, discard all the jars of the lot. Do not put explants in these jars.

Prepare the explant and sterilize its surface

In the first batches, use a stem piece with one bud, 10–15 mm (0.4–0.6 in) long. Do not use a meristem.

1. 1
   Mother

   The mother plant must be in vegetative growth. Examine it for pests. Use a new plant if you can. Use one cultivar.
2. 2
   Cut

   Cut in the morning. Each piece must be 10–15 mm (0.4–0.6 in) long. Remove the large leaves. Keep the pieces wet.
3. 3
   Clean with soap

   Use tap water with a drop of dish soap or Tween-20. Do this for 10–20 min.
4. 4
   70% alcohol

   Keep the pieces in the alcohol for 30–60 seconds. Then drain the alcohol.
5. 5
   Bleach

   Holmes used 10% household bleach (approximately 0.625% NaOCl) with 0.1% Tween-20 for 20 min. Mix the solution during this time.^[1] Das used 1% NaOCl for 30 min.^[4] Do not keep the pieces in the bleach for 60 min.
6. 6
   Clean with sterilized water

   Clean the pieces three times with sterilized water. Keep the pieces in the water for 3–5 min each time. Then put the pieces in the hood.
7. 7
   Remove the ends

   Cut the ends from the stem when they are white or have damage from the bleach. Put the new end of the stem in the gel.

Explants in jars: labels and incubation

Put one piece in each jar until you know your rate of good results.

1. 1
   Airflow

   Do the work 10–20 cm (4–8 in) in front of the filter. Do not do the work at the edge of the hood.
2. 2
   One lid

   Hold the lid with its face down, at the side of the jar. Do not hold the lid above the jar.
3. 3
   Put the explant in the jar

   Put the end that you cut in the gel. Keep the bud above the gel.
4. 4
   Lid

   Put the lid on the jar immediately. Do not speak above the jar.
5. 5
   Label

   Write the cultivar, the date, the type of explant and the lot number from F-03 on the label.
6. 6
   Shelf

   Keep the jars at 24–26 °C (75–79 °F). Use 16–18 h of light, at approximately 70–100 µmol m⁻² s⁻¹. Do not open a jar to examine the culture.

Examine the cultures and discard defective jars

Examine the jars on day 7 and on day 21. Look through the glass. Do not open a jar if you are not sure that it is clean.

Shoot multiplication and subculture

Use only jars that were clean on day 21. Use a new jar each time.

1. 1
   Source jar

   Use a jar that was clean on day 21. Use one cultivar.
2. 2
   Cut

   Cut a shoot tip or a node with a bud that you can see. Each piece must be 15–25 mm (0.6–1.0 in) long.
3. 3
   New jar

   Use the same medium or the medium that has no hormone and more calcium.^[4]
4. 4
   Density

   At home, put 1–3 explants in each jar. In a licensed facility, the rate in F-07 gives the number of explants for each jar.
5. 5
   Time

   The time for each subculture is 3–4 weeks. After approximately five subcultures, start the line again from a mother plant with a negative test result.^[9]

Meristem dissection after process validation

Use a microscope. The piece is 0.2–0.4 mm (0.008–0.016 in) long. Then do a lab test. This task does not make a plant “clean” if you do not do the lab test.

1. 1
   New growth

   Cut a vegetative tip of 10–15 mm (0.4–0.6 in). Remove the large leaves before you put the tip in the hood.
2. 2
   Sterilized tools

   Sterilize the tools in a glass-bead sterilizer at approximately 250 °C (approximately 480 °F) for 20 s. Then let the tools become cool.
3. 3
   Dish

   Put one drop of sterilized water on the dish. Use a magnification of 10–20× and then of 30–40×.
4. 4
   Remove the leaves

   Remove the outer leaves. Stop when you see two small leaves.
5. 5
   Put the tip in bleach

   Use a shorter time in the bleach than for a woody node. Then clean the tip with sterilized water.
6. 6
   One nick

   Make one nick. The piece must be 0.2–0.4 mm (0.008–0.016 in).^[6] Put the piece on a start medium of the Holmes type.^[1]
7. 7
   Wait

   Wait for 4–8 weeks. Then do the test on F-10. At six months, approximately 41% of the plants will have a negative test result. It is not 100%.

Rooting and acclimatization

A shoot with no roots is not a plant. In a culture jar, the relative humidity of the air is almost 100%. Thus the plant cannot close its stomata, and it does not make a layer of wax on its leaves. In the jar, it is not necessary for the plant to keep water.

When you move the plant directly to open air, it wilts in a small number of hours. The problem is the speed of the change and not the new conditions. Decrease the humidity in steps. This procedure is acclimatization.

1. 1
   Select a shoot

   Select a shoot of 2–4 cm (0.8–1.6 in). Do not select a shoot with glassy growth. Do not select a brown shoot.
2. 2
   Make roots

   Alternative A: put the shoot in gel with 5 µM IBA for 2–4 weeks.^[1] Alternative B: put the bottom end of the shoot in 15 mM IBA for 2–4 min. Then put the shoot in a sterilized plug.^[10] Alternative C: put the shoot on rockwool with usual fertilizer, in a jar with a vent and with no sugar.^[7]
3. 3
   Plug

   Use a plug of rockwool or coco. Soak the plug in a weak nutrient solution for vegetative growth. The pH must be approximately 5.8.
4. 4
   Dome

   Spray water on the walls of the dome. Use 16 h of light, with a low light intensity, at 24 °C (75 °F).
5. 5
   Vents

   On day 7, open the vents half. On day 9, open the vents fully. On day 14, remove the lid.
6. 6
   Pot

   Use the procedure for a new clone. For some weeks, do not use a photoperiod of 12 hours.

Tests, intake and lot records

If F-10 has no result, the plant is a clone. It is not a clean mother plant.

1. 1
   Sample

   Get a sample from a new leaf of full size, or from the leaf stalk. Do this after the plant makes new growth.
2. 2
   Lab

   Use a lab that does the RT-qPCR test for cannabis Hop latent viroid. Root tissue gives the most accurate result, if the lab can use roots.
3. 3
   Test again

   Do the test again 3–4 weeks after the first test, and before a mother plant goes into production.
4. 4
   Retain sample

   Freeze the remaining tissue from each lot with a “not detected” result.

Procedure of operation for each week

• After you do the procedure many times, you can do approximately 80–100 transfers each hour at a hood.^[15]
• Do the F-10 test on the production mother plants at intervals of 3–6 weeks.
• After approximately five subcultures, start a production line again from a backup with a negative test result.^[9]

Troubleshooting

Information on sources

The marks in this document point to this section. It is not necessary to read this section to do the work for one day.

The photos are illustrations. Use the diagrams for the size and the sequence.