This will be my first attempt at tissue culture propagation and preservation. Fail or succeed, here we go.
The current plan is to divide this experiment description into several sections:
Description of materials and equipment
Description of media formulation
Preparing the media for use and sterilization
Preparing for an aseptic (sterile) work area
Preparing plant tissue for culture initiation (stage I)
Preparing the tissue culture for multiplication (stage II)
Preparing the multiplied culture for rooting (stage III)
The documentation of this effort will take a little while, so please be patient.
The term “tissue culture” has been used as a catch-all phrase for the technique of using a sterile media along with a small amounts of plant material to propagate and multiply plant tissue in a vial or some sort of enclosed container.
The concept is based on the premise that a single plant cell containing a full copy of the plant DNA has sufficient information to regenerate an entire plant. This is called totipotency - the capacity of a cell (or a group of cells) to give rise to an entire organism.
Tissue culture, being an imprecise term, encompasses several techniques for differing reasons, including:
- Stem propagation, e.g. for cloning
- Micro propagation, e.g. for removal of microbiological contamination / infections using shoot-tips
- Meristem Culture, e.g. for removal of viruses using shoot-tip dissection (meristem)
- Protoplast Culture, e.g. undifferentiated cell culture for many engineering / research techniques
The process follows:
differentiated plant tissue (explant) dedifferentiation callus (heterogenous) redifferentiation (whole plant) cellular totipotency
By emulating different growth conditions and by manipulating the growth media, the tissue cells can be encouraged to regenerate an entire plant following the above process.
For instance, the addition of an auxin to the growth media encourages root development. Likewise, the addition of a cytokinen to the media encourages shoot development. Equal amounts of auxins and cytokinens encourages callus induction.
Auxin Cytokinen = root development
Auxin Cytokinen = shoot development
Auxin == Cytokinen = callus development
The most common use for tissue culture is micro-propagation but there are several other, namely scientific, uses including the ability to remove any viral or infectious load in the plant material.
The growth media itself has several characteristics:
- Inorganic salts consisting of macro and micro nutrients
- An organic source of carbon (sucrose, for instance)
Additional adjuncts may be added that have been shown to improve growth, such as:
- Amino acids
To provide support for plant growth, a gelling agent is added. Typically,
These comprise the base growing media. To this base growth media, plant auxins and cytokinens are added to encourage cell differentiation as described above.
The function of the culturing media, as a whole, is to:
- Provide water
- Provide mineral nutrition
- Provide vitamins
- Provide growth regulators
- Gas exchange
- Removal of plant metabolite waste
Materials and equipment
There are a variety documented methods, materials, formulations, and equipment required to successfully perform tissue culturing. To simplify sourcing the materials, I’ll be basing this trial off of the PhytoTechnologies “Hemp Multiplication Kit” with a couple of small modifications along with whatever additional equipment I have handy.
What are the materials?
Materials include the chemistry and disposable/use-once items.
- Sodium Bicarbonate
- MS Medium with calcium chloride and vitamins
- Distilled Water
- Beer and a bong
Vinegar. This is acetic acid. White distilled vinegar from the grocery store is fine. This will be used as a PH down.
Sodium Bicarbonate. This is also known as baking soda available from the grocery store. This will be used as a PH up.
Sucrose. This is also known as plain cane sugar also available from the grocery store.
Agar. Agar is a gelling medium. Alternative gelling mediums can be utilized.
IBA. This is an abbreviation that refers to Indole-3-butyric acid potassium salt (C12H12KNO2) or 4-[3-Indoly]Butyric Acid (C12H13NO2). It is a plant auxin precursor.
NAA. This is an abbreviation that refers to 1-Naphthaleneacetic acid. It is a naphthalene-based phytohormone plant auxin. This material is a skin irritant / sensitizer / and is corrosive.
TDZ. This is an abbreviation for Thidiazuron (C9H8N4OS). TDZ is a cytokinin plant growth regulator. It promotes promotes plant organogenesis (shoot regeneration) and plant regeneration. This material is a skin irritant / sensitizer / and is harmful to an aquatic environment.
MS Medium with calcium chloride and vitamins. MS refers to the Murashige & Skoog (1962) nutrient formulation. It is a common nutrient formulation available pre-made from a variety of sources. This formulation is suggested as being a good formulation for hemp / cannabis. If you want to formulate it your self (MS + calcium chloride + vitamins). The formulation is described later.
PPM (optional). This is an abbreviation for Plant Preservative Mixture. PPM is a commercial product that provides some antiseptic properties to the media that can be useful when preparing tissue cultures in the less than perfectly sterile conditions. When used in the proper proportions, PPM will not harm plant cell growth but will slow or eliminate bacterial / fungal growth.
Distilled Water. Don’t use tap water, use distilled.
Beer. Avoidance of parching.
- Culturing containers
- pH Strips or an accurate PH meter
- Bunsen Burner
- Balance or scale that is accurate to a minimum of 0.1g (100mg)
- Weigh boats
- Wash containers
- Laminar flow hood or sterile glove box
- Autoclave or pressure cooker
- Latex / Nitrile disposable gloves
- Sterilization Pouches (optional)
- Stir plate and bar (optional)
- Instrument rest (optional)
- Micropore tape (optional)
- Autoclave indicator tape (optional)
Culturing containers of some sort w/lid that can be autoclaved/sterilized (high temperature). Glass can be autoclaved. Polycarbonates and some Polypropylenes are tolerant up to 121 Celcius. Be sure to check whether the material can survive high temperatures. The following images are examples of potential containers. Left-to-right, flip-top containers, petri dish, rectangular culture container with removable lid.
Plastics that cannot handle the high heat of sterilization will not survive the process:
pH Strips or an accurate PH meter You’ll need some method to measure and verify the PH of the media. This can be using the typical techniques and materials as illustrated in the following image. From left-to-right, PH indicator drops, PH test strips, laboratory PH probe, PH pen. As usually, be sure things are set-up and calibrated ahead of time.
Basically, a really sharp knife, scissors, or a scalpel. Best being a scalpel. And, some sort of instrument to handle the plant material without damaging it or touching it with your hands. From left-to-right, scalpel blades, scalpel, forceps, tweezers.
A pipette is a device for accurately measuring generally small quantities of a liquid. From left-to-right and in order of precision, a “digital” micro-pipette, a glass pipette with pump, a plastic pipette, an “eye-dropper” with graduations.
Bunsen Burner or something for sterilizing instruments while handling plant material (such as a glass bead sterilizer). The following image illustrates an inexpensive bunsen burner that utilizes camping stove isobutane gas canisters. Alternatively, a high-proof alcohol may be sufficient in some circumstance although it may not be sufficient to immediately kill some types of spores.
Balance or scale that is accurate to a minimum of 0.1g (100mg). The following image details an analytical balance, you do not need something this precise. A scale like the ones used for reloading ammunition would be sufficient and are relatively inexpensive to purchase. Although, if you can find or afford a more precise scale / balance, they have many uses. If you plan on dealing with hormones and such, the quantities can be so small you’ll need as much additional precision as possible.
Weigh boats These are simply small containers you can use when measuring and weighing ingredients. The next image details some examples. It could be something a simple as a folded piece of paper.
Self explainatory. Have several available.
Laminar flow hood or sterile glove box
This will be described in detail later. A flow box or a sterile glove box are working locations that limits the contamination of your sterile media and equipment. Having a sterile work area (or as clean as possible) is essential. A localized working area that’s kept “extra” clean and free from contamination is what we are after.
Autoclave or pressure cooker This is used to sterilize the media, the containers, and any water that will be used for the tissue culture. The following image details an inexpensive 16-quart Presto brand pressure cooker. Notice this one has removable weights on the top of the cooker. The weight can be removed which will determine the cooking temperature when the unit is at pressure. For instance, 15psi weight will result in 121 Celsius / 250 Fahrenheit. A 10 psi weight results in 113 Celsius / 235 Fahrenheit. Etc.
Latex / Nitrile disposable gloves
Sterilization Pouches (optional)
Stir plate and stir bar (optional)
Instrument rest (optional) An instrument rest is simply a fixture that keeps the working parts of the scalpel, forceps, tweezers, etc off of the work surface. This is to avoid picking up any infectious contaminants that may have settled onto the work surface. Alternatively, placing the tools in a container filled with isopropyl alcohol would also be fine.
Micropore tape (optional)
Autoclave indicator tape (optional)
Formulations of the growth media vary depending on 1) the type of plant and 2) the individual theory 3) the goal. Similar to growing tomatoes versus oranges, the nutrient needs will vary. Past experimentation has shown what types of formulations work best for different plant species. As such, formulations are a guideline to what has worked in the past. This does not mean you, personally, shouldn’t vary a formulation. Experiment, succeed, improve.
The media formulation will also vary depending on the current goal. At different stages in the culture propagation, adjustments to the base formula with additional adjuncts will spur differing plant response.
The base media used here is called the Murashige & Skoog nutrient formulation. Vitamins and amino acids are added to this base formula. As noted earlier, this is a common formulation and is available on the commercial market pre-made. However, if you wish to make it yourself or modify the formula, here is one formulation:
1650ppm Ammonium Nitrate
332.2ppm Calcium Chloride, Anhydrous
1900ppm Potassium Nitrate
170ppm Potassium Phosphate, Monobasic
180.7 Magnesium Sulfate, Anhydrous
6.2ppm Boric Acid
0.025ppm Cobalt Chloride•6H2O
0.025ppm Cupric Sulfate•5H2O
27.8ppm Ferrous Sulfate•7H2O
16.9ppm Manganese Sulfate•H2O
0.25ppm Molybdic Acid (Sodium Salt)• 2H2O
0.83ppm Potassium Iodide
8.6ppm Zinc Sulfate•7H2O
0.5ppm Nicotinic Acid (Free Acid)
2.0ppm Glycine (Free Base)
Further information on the theory of nutrient formulation is referenced here: The Components of Plant Tissue Culture Media I.pdf (1.0 MB) and here: ComponentsOfTissueCultureMedia
Information of the theory of vitamin supplementation in culturing media is referenced here: The Components of Plant Tissue Culture Media ll.pdf (921.4 KB)
Sources for plant hormones:
Source for MS media with vitamins:
Other methods to find equipment:
Many of these items can also be found on Ebay for much cheaper cost-wise. Glassware, equipment, materials are often at significant discount. Used industrial items such as scales/balances can be found at pennies on the dollar if you know what to look for and are willing to entertain some risk. Like buying a used automobile. Scales and balances are sensitive to transport and are easily damaged, so be sure the vendor knows how to prep and pack the item for gorilla transport. The majority of the equipment items described above were sourced via Ebay. Industrial auctions are also a good source. Universities used to sell items as they are upgraded. I don’t know if they still do this but doesn’t hurt to ask. In each case, it just takes some time to find the good deals.
For chemistry purchased via Ebay, there will be some prep required and it is likewise somewhat difficult to verify the content and purity of chemistry purchased via Ebay. The amounts of hormones utilized at our scale is small so you don’t need much. Consider such tradeoffs if going through Ebay, as well.