Tissue Culture FAQ
What is Tissue Culture?
Tissue culture is a propogation method used to produce plants under sterile conditions. This method uses sterilized plant explants or seeds to produce plants. Many types of seeds germinated in these conditions tend to grow very fast compared to being sowed in standard growing media. From using plant tissue, it is possible to grow exact copies of the donor plant. This is extremely useful for plants that genetically have desirable traits.
The plants are grown in a nutrient media that contains a nutrient solution, sucrose (sugar), hormones (optional), antibiotics (optional), and a solidifying media.
Four stages of plant tissue culture have been defined by Murashige:
Stage I. Establishment of an aseptic culture.
Stage II. The multiplication of propagules.
Stage III. Preparation of propagules for successful transfer to soil (rooting and hardening).
Stage IV. Establishment in soil.
What is the difference between tissue culture and regular propogation by seeds, leaf pullings or flower stalks?
The main difference between tissue culture and normal growing is that the plants grow faster and you can multiply them rapidly by giving them the right hormones and dividing them regularly. In theory, you can create an infinite number of plants from one piece of tissue in a relatively short amount of time.
Here is a picture, courtesy of Jens, showing seeds started at the same time, in tissue culture, and in potting media.
What supplies do I need?
- Baby food jars (tall ones are better). You need lots of them. I got a ton of them for $20 off of Craigslist.
- Phyto Caps (go ahead and get 100 of them) for capping the baby food jars. They're autoclavable (can be put on the jars and put in a pressure cooker without exploding) and can be purchased from http://www.phytotechlab.com/
- Long forceps (at least 6 inches, 8 inches is better). You can order one from Carol if you can't find one in a store: http://www.kitchenculturekit.com/supplies-refills.aspx
- Media - Murashige and Skoog (MS) with vitamins
- A good pH meter. You can use paper strips, but they are very hard to get an accurate measurement with.
- A smidgen spoon measuring set
- A good pipette set or disposable glass pipettes.
- A pressure cooker.
- The basic stuff that you probably already have around the house including bleach, hydrogen peroxide, alcohol, sugar, distilled water, baking soda, vinegar and a teaspoon measuring set.
- Some sort of hood to work under. I use a 30 gallon aquarium laid on its side. Many people use a plastic storage bin, upside down with a hole cut out of the side. Laminar Flow Hoods are the ultimate work areas, but are somewhat expensive.
- A spray bottle to mist the inside of the work area.
- Any other hormones or chemicals that you want to work with. Many people use BAP for callus induction and IBA for root growth stimulation. I've read scholarly articles that say kinetin works great for callus induction on Dionaea. Make sure you decide on these before you place your order with Phytotech because they charge an arm and a leg for shipping, so you're better off not placing multiple orders with them. I recommend Plant Preservative Mixture (PPM). It helps a lot with sterilizing tissue and if you put it in the media, it helps prevent and control contamination. PPM can be ordered through Carol or directly from the PPM website: PPM for tissue culture
How do I prepare media?
If you order a kit from the Kitchen Culture Kits site by Carol Stiff, you'll get all the instructions you need for preparing media. Media preparation is somewhat dependent on the type of plant species you're attempting to put into tissue culture. Many species of plants can be cultured on full strength Murashige and Skoog (MS) with vitamins. For carnivorous plants, it's best to use 1/2 or 1/3 MS with vitamins. There are, of course, other types of media that could work as well, but for simplicity sake, let's just discuss using MS.
Here is a list of ingredients for preparing 1 liter of Dionaea muscipula medium:
- 1/2 MS medium with vitamins (one half of a 1 liter packet or 2.2 grams). Most Tissue Culture Techs uses 1/3 MS for Carnivorous plants with great results.
- 2 tablespoons of table sugar (about 30g)
- 1 ml of BAP (1 mg/ml) - leave this out for seeds. It's even optional for explants. Dionaea explants will form callus without it.
- 1 ml of PPM
- Distilled water
- Vinegar and baking soda.
- Fill a 1 quart or 1 liter container with 3 cups of water.
- Add MS medium (1/2 packet or 2.2 grams) and 2 tablespoons of table sugar and mix well.
- Measure 1 ml of BAP and 1ml of PPM and add them to the solution.
- Add food coloring (if desired) to indicate BAP media.
- Add enough water to bring the media mixture to exactly 1 liter
- Test the pH. A pH between 5 and 6 is preferred with 5.6 to 5.8 being optimal. [list]
- If the pH is too low ("acidic") add a VERY small amount of baking soda to the solution, remix it well and remeasure.
- If the pH is too high ("basic") add a few drops of vinegar to the solution, remix it well and remeasure.
- Continue doing this until you reach the desired pH.
[*]Add the agar to the jars. For 25 ml, I use a "smidgen" spoonful and for 50ml I use a "pinch" spoonful.[/*]
[*]Cap the jars and place them in the pressure cooker, microwave or other autoclave device in preparation for sterilization.[/*][/list]
Here is an excellent video tutorial for making media:
How do I sterilize media?
This stage involves treating the plantlets/shoots produced to encourage root growth and "hardening." It is performed in vitro, or in a sterile "test tube" environment.
Root growth does not always occur in the earlier stages in plant cell culture, and is of course a requirement for successful plant growth after the micropropagation procedure. It is often performed in vitro by transferring the plantlets to a growth medium containing auxin(s) which stimulate root initiation. The pretransplant stage is not always performed; Some plants are micropropagated and grown in culture and normal cuttings are made that are then rooted ex vitro.
"Hardening" refers to the preparation of the plants for a natural growth environment. Until this stage, the plantlets have been grown in "ideal" conditions, designed to encourage rapid growth. Due to lack of necessity, the plants are likely to be highly susceptible to disease and often do not have fully functional dermal coverings and will be inefficient in their use of water and energy. In vitro conditions are high in humidity and plants grown under these condition do not form a working cuticle and stomata that keep the plant from drying out, when taken out of culture the plantlets need time to adjust to more natural environmental conditions. Hardening typically involves slowly weaning the plantlets from a high-humidity, low light, warm environment to what would be considered a normal growth environment for the species in question. This is done by moving the plants to a location high in humidity, such as a green house with regular mist watering.
The key to sucessfully transferring your in-vitro plantlets into soil is to be very fastidious about washing off all the TC media from the roots.
Place the plantlet under running, tepid water, and use the force of the water to thoroughly dissolve off all the old media. If this is not done, then molds will inevitably take hold and overpower your plants.
After planting out, treat the plants the same as they were treated while still in-vitro. A humidity tent made with a zip-lock bag will help the plants acclimatize. Let them stay sealed for a week or so. You can then gradually open up the bag over the course of another week to get the plants used to lower humidity. Once they are "hardened off" properly, you can treat them as any other soil-grown plant.
What is Agar?
Agar is a gelatinous substance derived from seaweed. See here: http://en.wikipedia.org/wiki/Agar
It is primarily used for "firming" up media. You add it in powder form to the media, heat it to a boiling temperature to melt the agar, and then when the media cools back down, it "gels". The purpose of agar is to allow the explant or seeds not to sink into the liquid, but still have it soft enough that nutrients and water may be extracted from it.
How can I tell if my MS media already has agar in it?
You can figure this out by seeing how much the package tells you to add for 1L of media. If it is just the MS media, it will be ~4.33g/L, if it contains agar, it will be 9-12 g/L.
Do I need a flow hood?
Nope! Flow hoods are ideal, but they aren't necessary. Many people have had excellent results without the use of expensive flow hoods. Working in overturned plastic containers with the side cut out or aquariums laid on their side are also very good methods.
Can I build a flow hood?
Yes you can! It takes some good planning, and you need to source a couple of expensive items (HEPA filter, blower), but it is possible. See these threads for ideas:
How to build your own Laminar Flow Hood
Flow Hood Finished...Almost
Flow Hood - Build Thread
My flow hood
What are the colours I see in some peoples cultures?
These are simply food colouring added in to be able to distinguish different media types, or hormones added to the media. One exception to this is the addition of activated charcoal which will make the media a dark grey-black colour.
What are Plant Hormones?
How do I sterilize seeds?
Most species of carnivorous plant seeds are relatively easily sterilized. For first time tissue culturers, I would definitely recommend starting with seeds as explants can be very difficult to properly sterilize. One can hone their tissue culture skills with seeds and then move onto explants when they have a good sterile technique.
I use the same procedure for Dionaea and Sarracenia seeds (the only two species I've attempted) and it works really well.
- Quick dip in 91% ethanol alcohol.
- 2 to 3 minutes in 3% Hydrogen Peroxide
- 10 minutes in 10% bleach
- 3 minute rinse in sterile water
- Transfer to media - I use forceps to pick up each individual seed. A wire ring works well too.
How do I sterilize explants?
I alter the exposure times to the sterilization chemicals depending on the size of the tissue. If the tissue is larger or thicker (like stalks) I let it soak longer. If it's thin or small (like most leaves) I decrease the soak time. So it's more of an art than a hard science when it comes to sterilizing tissue. However, here are the basic steps of my sterilization technique:
- Harvest the cleanest, youngest piece of tissue I can. I've been growing any plants that I want to put into TC indoors to help keep them clean.
- Thoroughly rinse the tissue under warm running water for 20 to 30 minutes. I usually start the wash with antibacterial soap that has triclosan in it (either Dial complete or a similar generic brand). Then after some time (15 minutes?) washing/soaking in the soapy water, I just run it under water.
- Quick dip in 91% alcohol. For small pieces of tissue I skip this step because it seems to me that alcohol can quickly kill the tissue.
- Transfer to 3% H2O2. For small tissue, leave it in for 3 to 5 minutes. Larger tissue, 5 to 7 minutes. I shake the container vigorously during this time.
- Transfer to 200ml of 10% to 20% bleach (I change it between 10%, 15% and 20% and it doesn't seem to make much of a difference) with 6 to 8 drops of Tween 20. Again, the amount of time in the bleach varies depending on the size of the tissue, but it usually ranges from 6 minutes to 12 minutes. I shake the container vigorously during this time.
- Two 3 to 5 minute rinses in sterile water and then transfer to media.
How do I make 10% Bleach?
For making small amounts, mix 10 ml of bleach to 90 ml distilled/RO water.
How do I make 3% Hydrogen Peroxide?
3% Hydrogen Peroxide is readily available in Pharmacies and Drug stores.
Do I have to use PPM?
You can either use PPM or make a media without it. You get less chance of contamination if you use PPM.
What is a callus?
A callus of cells is a mass of undifferentiated plant cells.
What is activated charcoal?
Charcoal which has been treated to remove hydrocarbons and to increase its adsorptive properties. It acts by condensing and holding a gas or solute onto its surface; thus inhibitory substances in nutrient medium may be adsorbed to charcoal included in the medium. Rooting factors such as phenolamines present as contaminants in charcoal may stimulate growth in vitro. Its addition to rooting medium may stimulate root initiation in some plant species. Activated charcoal may differ in origin and in composition. cf charcoal; phenolic exudation.
How long does it take to see callus formation?
It all depends on the species of plants and which part you use for the explant. The latest is 6 weeks.
Tips & Tricks
Abaxial - On the side that is away from the axis or central line, usually on the underside of the explant - See here.
Abscisic Acid - ABA. A plant growth regulator involved in abscission, dormancy, stomatal opening/closure, and inhibition of seed germination. It also affects the regulation of somatic cell embryogenesis in some plant species.
Activated Charcoal - Charcoal which has been treated to remove hydrocarbons and to increase its adsorptive properties
Adaxial - On the side that is towards the axis or central line, usually on the upper side of the explant - See here.
Adventitious Bud - Buds that develop in places other that at the end of a twig or in leaf axils. They appear when wounding stimulates their development.
Agar - A polysaccharide solidifying agent used in nutrient media preparations and obtained from certain types of red algae (Rhodophyta). Both the type of agar and its concentration can affect the growth and appearance of cultured explants
Antibiotic - Any chemical or biological agent that harms the growth of micro-organisms.
Apomixis - Replacement of the normal sexual reproduction by asexual reproduction, without fertilization. Thus "normal asexual reproduction" of plants, such as propagation from cuttings or leaves, has never been considered to be apomixis, but replacement of the seed by a plantlet, or replacement of the flower by bulbils are types of apomixis. Apomictically produced offspring are genetically identical to the parent plant.
Aseptic - The state of being free of contaminating organisms (bacteria, fungi, algae and all micro-organisms except viruses). See Sterile.
Autoclave - 1. An enclosed chamber in which substances are heated under pressure to sterilize utensils, liquids, glassware, etc., using steam. The routine method uses steam pressure of 103.4×103 Pa at 121°C for 15 minutes, or longer to allow large volumes to reach the critical temperature.
2. A pressure cooker used to sterilize growth medium and instruments for tissue culture work.
Auxin - A group of plant growth regulators (natural or synthetic) which stimulate cell division, enlargement, apical dominance, root initiation, and flowering.
Axillary bud - when located in the axil of a leaf (lateral is equivalent but some adventitious buds may be lateral too)
Bleach - A fluid, powder or other whitening (bleaching) or cleaning agent, usually with free chlorine ions. Commercial bleach contains calcium hypochlorite or sodium hypochlorite, and is a common disinfectant used for cleaning working surfaces, tools and plant materials in plant tissue culture and grafting.
Bleeding - Used to describe the occasional purplish-black coloration of media due to phenolic products given off by (usually fresh) transfers.
Bud - An undeveloped or embryonic shoot and normally occurs in the axil of a leaf or at the tip of the stem. Once formed, a bud may remain for some time in a dormant condition, or it may form a shoot immediately.
Callus - A mass of thin-walled, undifferentiated plant cells, developed as the result of culture on nutrient media.
Cell Differentiation - Continuous loss of physiological and cytological characters of young cells, resulting in getting the characters of adult cells. The unspecialized cells become modified and specialized for the performance of specific functions. Differentiation results from the controlled activation and de-activation of genes.
Charcoal - See Activated Charcoal
Clone - Group of plants genetically identical in which all are derived from one selected individual by vegetative or in-vitro propagation, without the sexual process.
Cultivar - A category of plants that are, firstly, below the level of a sub-species taxonomically, and, secondly, found only in cultivation. It is an international term denoting certain cultivated plants that are clearly distinguishable from others by stated characteristics and that retain their distinguishing characters when reproduced under specific conditions.
Culture Medium - See Medium
Cutting - A detached plant part that under appropriate cultural conditions can regenerate the complete plant without a sexual process.
Cytokinin - Plant growth regulators (hormones) characterized as substances that induce cell division and cell differentiation (e.g., BAP, kinetin, and 2-iP). In tissue culture, these substances are associated with enhanced callus and shoot development.
Differentiation - A process in which unspecialized cells develop structures and functions characteristic of a particular type of cell. Development from one cell to many cells, accompanied by a modification of the new cells for the performance of particular functions. In tissue culture, the term is used to describe the formation of different cell types.
Disinfectation - Full elimination of internal micro-organisms from a culture.
Distal - The side of an explant furthest from the point of attachment (i.e. the tip of a leaf)
Donor Plant - An explant, graft or cutting used as a source of plant material for micro-propagation purposes.
Established Culture - An aseptic viable explant.
Ethanol - C2H6O, Commonly used to disinfest plant tissues, glassware utensils and working surfaces in tissue culture manipulations. The concentration used is 70% (v/v) for disinfecting and 95% (v/v) when flaming tools.
Excision - Cutting out and preparing a tissue, organ, etc., for culture.
Explant - Tissue asceptically obtained and prepared from the donor plant for culture(flower stalk, leaves, roots)
Exponential phase - See Logarithmic phase
Ex Vitro - Organisms removed from tissue culture and transplanted; generally plants to soil or potting mixture.
Filter Sterilization - Process of sterilizing a liquid by passage through a filter with pores so small that they prevent the passage of micro-organisms and microbial spores.
Flaming - A technique for sterilizing instruments. The instrument is dipped in alcohol (usually 95% (v/v) ethanol) and then the alcohol on the instrument is ignited, thus heat-sterilizing the tool surface.
Formulation - See Medium
Fungicide - An agent, such as a chemical, that kills fungi.
Gelatin - A glutinous, proteinaceous gelling and solidifying agent. Occasionally used in place of agar in tissue culture.
Giberellins - Plant growth regulators involved in elongation, enhancement of flower, fruit and leaf size, germination, vernalization and other processes.
Hardening off - Adapting plants to outdoor conditions by gradually withholding water, lowering the temperature, increasing light intensity, or reducing the nutrient supply. The hardening-off process conditions plants for survival when transplanted outdoors. The term is also used for gradual acclimatization to in vivo conditions of plants grown in vitro, e.g., gradual decrease in humidity. cf acclimatization; free-living conditions.
HEPA Filter - (high efficiency particulate air filter) A filter capable of screening out particles larger than 0.3 µm. HEPA filters are used in laminar air flow cabinets (hoods) for sterile transfer work.
Hormone - A specific organic product, produced in one part of a plant, and transported to another part where, at low concentrations, it promotes, inhibits or quantitatively modifies a biological process.
Hypochlorite - Generic term for aqueous solutions of sodium hypochlorite, potassium hypochlorite or calcium hypochlorite, which are oxidizing agents and used for disinfecting surfaces and surface-sterilizing tissues, and for bleaching.
Initiation - Early steps or stages of a tissue culture process (culture growth, organogenesis, embryogenesis)
Inoculum - A small piece of tissue cut from callus, or an explant from a tissue transferred into fresh medium for continued growth of the culture.
Inositol - C6H6(OH)6, A water-soluble nutrient frequently referred to as a "vitamin" in plant tissue culture.
In vitro - Living in test tubes, outside the organism or in an artificial environment, typically in glass vessels in which cultured cells, tissues, or whole plants may reside.
In vivo - The natural conditions in which organisms reside. Refers to biological processes that take place within a living organism or cell under normal conditions.
Kinetin - One of the cytokinins, a group of growth regulators that characteristically promote cell division in plants.
Kinin - A substance promoting cell division. In plant systems, the prefix cyto- has been added (cytokinin) to distinguish it from kinin in animal systems. See cytokinin.
Lag phase - The initial growth phase after inoculation, during which cell number remains relatively constant, prior to rapid growth.
Laminar Flow Hood - Cabinet for inoculation of cultures. The working area is kept sterile by a continuous, non-turbulent flow of sterilized air through a HEPA filter. See HEPA filter
Log phase - See Logarithmic phase
Logarithmic phase - The steepest slope of the growth curve; the phase of vigorous growth, during which cell number doubles every 20-30 minutes.
Longitudinal edge - The direction parallel to the ridge, following the greatest length of an area or object.
Macronutrient - For growth media: an essential element normally required in concentrations >0.5 millimole/l.
Media - See Medium
Medium - In plant tissue culture, a term for the liquid or solidified formulation upon which plant cells, tissues or organs develop.
Medium Formulation - Medium formulation In tissue culture, the particular formula for the culture medium. It commonly contains macro-elements and micro-elements , some vitamins (B vitamins, inositol), plant growth regulators (auxin, cytokinin and sometimes gibberellin), a carbohydrate source (usually sucrose or glucose) and often other substances, such as amino acids or complex growth factors. Media may be liquid or solidified with agar; the pH is adjusted (ca. 5-6) and the solution is sterilized (usually by filtration or autoclaving). Some formulations are very specific in the kind of explant or plant species that can be maintained; some are very general.
Meristem - Undifferentiated tissue, the cells of which are capable of active cell division and differentiation into specialized and permanent tissue such as shoots and roots.
Micronutrient - For growth media: An essential element normally required in concentrations < 0.5 millimole/litre.
Mother plant - See Donor plant
Myo Inositol - See Inositol
MS - Murashige and Skoog media
Nutrient medium - See Medium
Organized tissue - Composed of regularly differentiated cells.
pH - A measure of acidity and alkalinity. Equal to the log of the reciprocal of the hydrogen ion concentration of a solution, expressed in grams per litre. A reading of 7 is neutral (e.g., pure water), whereas below 7 is acid and above 7 is alkaline.
Phenolic Exudation - Many plant species contain phenolic compounds that blacken through exudation. The process is initiated after plants are wounded. Phenolic exudation may lead to growth inhibition or, in severe cases, to tissue necrosis and death. Antioxidants are incorporated into the sterilizing solution or isolation medium to prevent or reduce exudative browning (activated charcoal).
PPM - Plant Preservative Mixture - a broad-spectrum biocide/fungicide for plant tissue culture.
Propagule - Any structure capable of giving rise to a new plant by asexual or sexual reproduction, including seeds, leaves, buds, etc.
Proximal - The side of an explant closest to the point of attachment (i.e. the base of a leaf)
Radicle - That portion of the plant embryo which develops into the primary or seed root.
Scarification - The chemical or physical treatment given to some seeds (where the seed coats are very hard or contain germination inhibitors) in order to break or weaken the seed coat sufficiently to permit germination.
Semi-solid - Gelled but not firmly so; small amounts of a gelling agent are used to obtain a semi-solid medium; called also semi-liquid.
Spore - A small, protected reproductive form of a micro-organism, often synthesized when nutrient levels are low.
Stationary phase - The plateau of the growth curve after log growth, during which cell number remains constant. New cells are produced at the same rate as older cells die.
Sterile - Medium or object with no perceptible or viable micro-organisms.
Sterilize - The process of elimination of micro-organisms, such as by chemicals, heat, irradiation or filtration.
Sub-culture - Division and transfer of a portion or inoculum of a culture to fresh medium. Sometimes used to denote the adding of fresh liquid to a suspension culture.
Terminal bud - when located at the tip of a stem (apical is equivalent but rather reserved for the one at the top of the plant)
Tissue culture - A general term used to describe the culture of cells, tissues or organs in a nutrient medium under sterile conditions
Transverse - Across the width of the explant, the direction perpendicular to the ridge, the perpendicular side of the longitudinal side.
Undefined medium - A medium or substance added to medium in which not all of the constituents or their concentrations are chemically defined, such as media containing coconut milk, malt extract, casein hydrolysate, fish emulsion or other complex compounds.
Undifferentiated - Lacking the specialized or differential gene expression characteristic of specialized cells.
Vessel - A container, such as a Petri dish, jar, baby food jar or test tube, used for tissue culture.
Viability - The capability to live and develop normally.
Viable - Capable of germinating, living, growing and developing.
w/v - Weight per volume; the weight of a constituent in 100 cm3 of solution, expressed as a percentage.
Zone of elongation - The section of the young root or shoot just behind the apical meristem, in which the cells are enlarging and elongating rapidly.
Majority of glossary terms sourced from: http://www.fao.org FAO RESEARCH AND TECHNOLOGY PAPER No. 7