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Tissue Culture

I’m in the process of building a tissue culture lab and would love some direction on best practices and SOPs. This is not my wheelhouse and I could use some direction on what I should incorporate into this build. We have a commercial kitchen in a trailer that’s a little under 400 square feet. We are setting up the autoclave and clean room. I’m in Tucson and would love to see an operating tissue culture lab and I would be willing to travel. Any direction is appreciated.

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There may be a member or two from the @LabOwner, @LabEmployees, @Geneticists, @mastergrowers, @growopowners or @GrowOpEmployees who can lend a hand here with some TC pointers. Can somebody please help out @mdrust with some tissue culture info and maybe a facility tour?

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Micropropagation creates soma clones used to reproduce high yielding, diseased plants can recovered from meristem. fast producing plants. you will need the necessary environment and essential ingredients.

Sucrose, inorganic salts, vitamins, amino acids, growth regulators (auxins), and cytokinins.

This creates a callus from the tissue that is unorganized. Multiplied and transferred too containers for rapid growth.

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@mdrust I can share some pointers for you. Let me sync up with the PhytoTechnology team and see if we can outline some direction for you. I’ll get back ASAP!

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I just looked at the phototechnology laboratory website…good stuff. I’ll look forward to learning more. Thank you,

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If you haven’t read the half dozen or so published articles on Cannabis tissue culture, I suggest you head over to Google scholar and check those out. The number one piece of advice I would offer is, always be experimenting with recipes. The basal hormone levels of Cannabis cultivars are all over the map, requiring nearly cultivar specific recipes at each step of the process. Definitely consider each plants tendencies when coming up with recipes. For instance, does cultivar A root faster than any other clone you have? If so, maybe cut back on the root hormones in the recipe and see if that helps, etc.

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@mdrust I had a chance to sync up with the Phyto team and we came up with the following for you:

  1. I’d like to point you toward Phyto’s Explant Decontamination (Prod. No. E2650) manual as it will provide good general practices for working aseptically with tissue in culture.

E2650 manual link: https://phytotechlab.com/mwdownloads/download/link/id/3932/

  1. Another good source of information is their Hemp kit for media preparation:

C1850 manual link: https://phytotechlab.com/mwdownloads/download/link/id/2343/

  1. The book “Plants from Test Tubes” by Lydiane Kyte also provides a chapter dedicated to designing a laboratory - just FYI!

  2. An autoclave, pH meter, deionized water, and a horizontal laminar flow hood are what we generally recommend to prepare media and perform subcultures initially.

In addition to this, Phyto will have an 8 hr. Short-course they will be announcing soon where they’ll essentially teach a primer on plant tissue culture out here in California this summer, and if this is received well, they are looking to provide an extended course (2+ days) in a laboratory. Once this becomes more concrete, I’ll certainly share the brochure with everyone here.

I hope this is helpful, please let me know if you have any questions.

Brice

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The Phytotech website has tons of information. I’m definitely interested in the short course this summer, so keep me posted. I will look into the other materials you recommended. Thank you.

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It’s a huge industry with lots of research money being spent on it, especially

when it’s combined with gene research.

See below for some important thoughts on the topic.

Research organizations, specifically the National Research Council Canada.

https://www.nrc-cnrc.gc.ca/eng/solutions/advisory/cell_tech_tissue_culture.html

are dedicated to research.

Other organizations include botanical gardens and universities to name a few.

One of the benefits of micropropagation is the high number of plants that

can be produced.

However because plants are sub-cultured and divided hundreds or thousands

of times, contamination errors can quickly multiply and add up to great

losses. Therefore when designing a laboratory for propagation purposes, no

matter the size, there are certain criteria necessary to ensure the sterility and

environmental control necessary for successful production of clean, high

quality plants.

Location

A suitable location for a lab will be isolated from traffic, have restricted access,

thermostatically controlled heat, potable water and drainage, adequate electrical circuitry,

compatibility for fan and intake blowers and good lighting.

Depending on the size of the operation, physical isolation by building a free-standing

structure will help with sanitary upkeep and foot traffic.

Facilities

A sealed concrete floor, insulated and water resistant walls and ceilings are required. A

system for heating and cooling should be included to ensure constant environmental conditions

in peak seasons. Potable water and drainage system is required.

General Design

Cleanliness is one of the most important considerations in designing a tissue culture lab.

Common to all tissue culture facilities are: Washing equipment and storage areas, media

preparation, sterilization and storage areas, transfer areas for aseptic processing, incubation

areas with climate control and observation areas for data collection.

Routine cleaning and aseptic standard operating procedures can help eliminate losses.

All surfaces should be easy and efficient to wash. High efficiency particulate air (HEPA) filters

installed on HVAC intakes can reduce outside contaminats. Entrances

should have sticky mats, shoe covers or foot baths to reduce dirt entering the facility.

The lab layout should maximize cleanliness during workflow.

Glassware Washing and Storage Area

This area should be close to the sterilization and media preparation areas. Washing and

storage areas contain sinks, washing machines, drying ovens, autoclaves, sterilized water, dustproof

cupboards and waste disposal.

Media Preparation, Sterilization and Storage Area

This bench area will contain equipment. Scales are used to ensure proper weights of

substrate components. pH meters are present to measure media pH. Hot plate stirrers are used

to make semi-solid media to eliminate having to do it manually over a stove-top. Aspirators or

vacuum pumps can be added to a water source for filter sterilization of chemicals and to

disinfect plant material. Autoclaves use high heat to sterilize media, water, glassware and

utensils. Microwave ovens may expedite defrosting frozen media and heating agar. Dissecting

microscopes will be required for meristem propagation, dissection of floral or shoot apices and

general monitoring. This area may also include media dispensors,

preparation vessels and refrigerated storage.

Aseptic Transfer Area

The aseptic transfer area is where the plants are physically handled and transferred into

or out of culturing vessels, typically under laminar flow hoods.

Equipment required includes forceps, scalpels, equipment stands, labeling pens and sanitizing

equipment such as sterilizers, alcohol, Bunsen burners or alcohol lamps. Entrance to this room should

be restricted and entrants should use shoe covers, lab coats or coveralls. Smooth surfaces prevent

dust accumulation and UV lamps may be used for sterilization.

Primary Growth Room

Along with cleanliness, primary growth rooms are designed to maximize environmental

control with respect to temperature, relative humidity, air circulation and lighting.

Observation and Data Collection Area

A portion or room of the tissue culture lab should include an area in which plant analysis

can be performed and recorded. A designated space enables data collection for records for:

Media protocols, plant inventory and production records, shipping schedules, audit results,

nuclear stock certificates, testing results, phytosanitary and propagation import permits and

inventory records. It is the brain of the tissue culture lab.

I would say Tissue Culture is here to stay as the value lies in both as a means of increasing the

numbers of critically important plants for use around the globe and for furthering human

understanding and knowledge of flora.

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Thank you for taking the time to share this information. I went through my checklist to make sure I didn’t miss anything. Most of all it i feel even more focused on keeping clean. We are setting up a clean room (prelab) with air doors and ventilation hood to keep contamination out of the lab. Our space was a commercial kitchen and has clean walls and ceiling but after your post I want to look into sealing our floors and adding insulation where I can. Thank you again for your input.

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No worries. Feel free to PM me what you got. I would love to look over your design.

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It’s cool that you are setting up a TC lab, @mdrust! Is it here in town? I’d love to come document some of your process along the way, if you’d be down.

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