Nutrient Media for Plant Tissue Cultures
One of the first decisions that must be made when developing a tissue culture system is what
medium to use. Nutrient media for plant tissue culture are designed to allow plant tissues to be
maintained in a totally artificial environment. Many different tissue culture media have been
developed, but only a few have found wide-spread use, e.g. MS (Murashige and Skoog, 1962). SH
(Shenck and Hildebrandt), and Gamborg's B5. One of the most successful media, devised by
Murashige and Skoog (Murashige and Skoog, 1962) was formulated by analyzing the inorganic
components in tobacco plants and then adding them to medium in amounts similar to those found
in the plants. Not only did they find that the ions themselves were important, but the forms in
which the ions were supplied were critical as well.
In addition to mineral elements, the macro- and micronutrients that are similar to what is found in
fertilizers, nutrient media also contain organic compounds such as vitamins, plant growth regulators,
and a carbon source.
I. Mineral elements
A. Macroelements consist of N, K, P, Ca, Mg, and S.
1. Nitrogen (N) - Nitrogen is essential for plant growth. Most inorganic nitrogen is converted to
amino acids and then to proteins. Nitrogen is typically added to plant nutrient media as the nitrate
ion (NO3-, oxidized) and/or the ammonium ion (NH4+, reduced), which are added as inorganic salts.
Inorganic nitrogen generally ranges from 25-60 mM in nutrient media. In devising media, both the
total amount of nitrogen as well as the relative amounts of NO3- and NH4+ are important. There are
usually lower levels of NH4+ than NO3- in medium; nitrate is usually added at concentrations
between 25 and 20 mM and ammonium at concentrations between 2 and 20 mM. For example, the
amount of NH4+ in MS medium is less than half that of NO3- and in other media the NH4+
concentration is lower still. Cultures of some species can proliferate on medium containing nitrates
alone, and some can grow on a medium with ammonium as the sole inorganic nitrogen source if one
or more of the TCA cycle acids (citrate, succinate, malate) are included in the medium at
concentrations of about 10 mM. In poorly buffered media, the use of both nitrogen forms helps
maintain pH. Also, many plant species appear to respond best if they are given both forms, although
the reason for this is not known.
Nitrogen may also be added to medium in an organic form, as amino acids such as proline or
glutamine, hydrolysates (such as casein hydrolysate), or, as above, as organic acids. Organic nitrogen
is already reduced, i.e. in the form in which most nitrogen exists in the plant, and so may be taken up
more readily than inorganic nitrogen. The organic forms are often added to media that do not
contain ammonium. However, almost always, some inorganic nitrogen is present.
2. Potassium (K) - Potassium is the major ion in plants with a positive charge, balancing negative
ions. Although the amount of potassium required varies widely among different species, in media
potassium concentration is generally correlated with that of nitrate and ranges between 20-30 mM.
3. Phosphorous (P) - Phosphorus is an integral part of nucleic acids and other structural
compounds. It is added to culture medium as phosphate (PO4-) in sodium or potassium hydrogen
phosphates in concentrations ranging from 1-3 mM.
4. Calcium (Ca) - Calcium is a co-factor of many enzymes and is particularly important in cell wall
synthesis. It is supplied mostly as calcium chloride or calcium nitrate, concentrations ranging
between 1 and 3 mM. In plant cultures, calcium deficiency may result in shoot tip necrosis.
5. Magnesium (Mg) - Magnesium is critical for the functioning of enzymes, is an integral
component of the chlorophyll molecule, and is a cation that balances negative ions. It is usually
added as magnesium sulfate in concentrations similar to that of calcium.
6. Sulfur (S) - Sulfur is a part of several amino acids and has an important function in protein
structure. It is supplied as the SO4- ion, generally with magnesium as the cation, in concentrations
ranging from 1-3 mM.
B. Micronutrients
Micronutrients used in plant tissue culture are Fe, Mn, Zn, B, Cu and Mo, Co, and I.
1. Iron (Fe) - Iron is necessary for chlorophyll synthesis and functions in many oxidation/reduction
reactions. It is generally present in media at approximately 1 M. The major problem in supplying
iron in vitro is that it forms insoluble compounds in alkaline pH, a problem that is particularly
evident in liquid culture, where it may be seen as a precipitate. The use of chelating agents, which
bind metal ions, makes iron more stable and available to plant tissues over wider pH ranges.
Although there are several of these, the sodium or potassium form of ethylenediaminetetraacetic
acid (EDTA) is most often used because it is not as toxic as other chelating agents and it enables
iron to be available to cultures over a wider pH range than other agents. Fe-EDTA may be
purchased as a salt or prepared from ferric sulfate and EDTA.
2. Manganese (Mn) - Manganese is required for enzyme reactions, particularly in respiratory and
photosynthetic processes and is usually added as manganese sulfate in concentrations of 5-30 M.
3. Zinc (Zn) - Zinc is also required in many enzyme activities and is added to medium in
concentrations similar to that of manganese. The most common form in which zinc is added is as
the sulfate salt.
4. Boron (B) - Boron is an essential element involved in lignin biosynthesis and metabolism of
phenolic acids and is supplied as boric acid in culture medium (25-100 M). Boron deficiency results
in the death of shoot tip meristems.
5. Copper (Cu) - Copper is critical in many enzyme reactions, including the cytochrome oxidase
system. It is added to culture medium (as cupric sulfate) in very low concentrations (0.1 M),
because high amounts can be toxic.
6. Molybdenum (Mo) - Molybdenum functions in the transformation of nitrate to ammonium. It is
added as sodium molybdate in low concentrations (1 M) in culture medium.
7. Cobalt (Co) - Cobalt is not considered to be an essential mineral by plant physiologists, but is
included in many of the most widely used media formulations. Cobalt is supplied in concentrations
similar to that of copper, again because it may be toxic at higher concentrations.
8. Iodine (I) - Iodine is not considered to be an essential element, but it is often added to plant
culture media (5 M) because it has been found to improve growth of roots and callus in vitro.
9. Other elements - Nickel (Ni), aluminum (Al), and silicon (Si), are added to a few media
formulations. These elements have not been found to be necessary for most plant species in vitro.
II. Organic Compounds
Organic compounds are also added to plant culture medium. Some of these compounds, such as
sugars, are absolutely needed for growth, while others, such as vitamins, undefined compounds, and
organic acids, may not be essential but may enhance growth.
A. Sugars
Most plant tissue cultures are not highly autotrophic, that is, capable of fixing carbon through
photosynthesis, due to limitations in culture of CO2 availability, among other factors. Therefore,
sugar is added to the medium as an energy source. Sucrose is the most common sugar added,
although glucose, fructose, and sorbitol are also used in certain instances. Sucrose is the sugar form
most commonly transported in plants; it is broken down into glucose and fructose during
metabolism. It is also partially hydrolyzed into glucose and fructose during autoclaving. The
concentration of sugars in nutrient media generally ranges from 20 to 40 g/l.
Sugars also act as an osmoticum in the medium. Osmotic potential can have an important effect on
in vitro response. Nutrient salts contribute from 20% to 50% of the osmotic potential of media,
with sucrose making up the rest. When sucrose is hydrolyzed, as during autoclaving, its contribution
to the osmotic potential is further increased.
B. Vitamins
Only thiamine (vitamin B1), which is required for carbohydrate metabolism and the biosynthesis of
some amino acids, has been shown to be essential for most plant cultures. Nicotinic acid (niacin)
and pyridoxine (B6) are also commonly added to Murashige and Skoog medium and some other
media. Other vitamins such as biotin, folic acid, ascorbic acid (vitamin C), and vitamin E
(tocopherol) are sometimes added to media formulations. Vitamin concentrations are generally very
low.
C. Myo-inositol
Myo-inositol, a sugar alcohol, is added to most plant culture media. Although not essential for
culture viability, it can significantly improve in vitro response, especially in monocots. Although
myo-inositol is not essential for growth of many plant species, its effect on growth is significant.
D. Complex organics
These are undefined supplements such as coconut milk, coconut water, yeast extract, fruit juices and
fruit pulps. They may supply amino acids, vitamins, plant growth regulators, and/or secondary plant
metabolites. Complex organics were frequently used early in the history of plant tissue culture, when
growth requirements were less defined. Now they are used only when no combination of defined
components supports growth. Their disadvantages are that the important compounds in them are
not known, and may vary greatly from batch to batch. Only protein hydrolysates and coconut milk
(at 5-20% v/v) are used much today.
III. Activated Charcoal
Activated charcoal is sometimes added to media in order to adsorb toxic compounds released by
plant tissues, particularly oxidized phenolics. It may be especially useful in rooting medium.
However, activated charcoal adsorbs not only toxic compounds, but also growth regulators and
other compounds that are added to the medium. Activated charcoal is usually acid-washed prior to
addition to the culture medium at a concentration of 0.5-3.0 %.
IV. Solidifying Agents
Solidifying agents are used to create semi-solid or solid media wherein plant cultures are not
submerged in the medium. Liquid medium can be used for many plants but it must usually be
agitated to provide sufficient oxygen to the tissue.
A. Agar
Agar is the most commonly used gelling agent. Marine red algae contain the structural
polysaccharide agar, which consists of 2 components, agarose and agaropectin. Agarose is
composed of alternating D-galactose and 3,6-anhydro-L-galactose with side chains of 6-methyl-Dgalactose residues. Agaropectin is like agarose but additionally contains sulfate ester side chains and
D-glucuronic acid. The tertiary structure of agarose is a double helix with a so-called threefold
screw axis. The central cavity of this double helix can accommodate H2O molecules. Agarose and
agaropectin readily form gels that contain high amounts of H2O (up to 99.5%). When agar is mixed
with liquid, it forms a gel that melts at about 100 C and solidifies at about 45 C. Other benefits are
that agar does not react with any components of the medium and it is not digested by enzymes from
the plant tissue. All agar contains impurities, such as inorganic salts, organic compounds, phenolics,
and long chain fatty acids; amounts and types vary depending on the manufacturer. These
compounds usually do not interfere with culture response. If necessary, agar can be washed to
remove inhibitory impurities. Agar does not gel well under acidic conditions (pH <4.5). The
inclusion of activated charcoal in media may also inhibit gelling of agar. The agar concentrations
commonly used in plant culture media range between 0.5% and 1%; these concentrations yield a
firm gel at typical media pHs. The concentration of agar may be critical to plant response in culture.
Medium that is too soft may produce hyperhydric (abnormal, glassy-looking) tissue while medium
that is too hard may cause reduced growth.
B. Agarose
When greater purity is needed, agarose may be used. Agarose is extracted from agar leaving behind
agaropectin and its sulfate groups. Because of the additional purification, agarose is considerably
more expensive than agar. Agarose also has higher gel strength than agar and thus less is required for
solidification of media. Agarose is used in situations where the impurities of agar are a major
Disadvantage, such as in protoplast culture.
C. Gelrite
Gelrite™ consists of a polysaccharide produced by the bacterium Pseudomonas elodea. Medium
solidified with Gelrite has the advantage of being clear, which agar-solidified medium is not.
Consequently contamination is more easily detected at an early stage. Impurities in Gelrite contain
inorganic ions, but no organic compounds. Gelrite requires more stirring than agar when being
added to media. Unlike agar, Gelrite cannot be reheated and gelled successfully. One limitation of
Gelrite is that the concentration of divalent cations such as calcium and magnesium ions must be
within the range of 4-8 mM/liter. Concentrations of these two ions either less than or greater than
this range result in the media not gelling. Gelrite™ may also produce hyperhydric plants when used
at low concentrations.
D. Phytagel
Phytagel™ is an agar substitute produced from a bacterial substrate composed of glucuronic acid,
rhamnose and glucose. It produces a clear, colorless, high-strength gel, which aids in detection of
microbial contamination. Phytagel provides an economical alternative to agar as a gelling agent. It is
used at a concentration of 1.5-2.5 g/L. To prevent clumping, Phytagel should be added to rapidly
stirring culture medium which is at room temperature. Hyperhydricity may also be a problem with
this gelling agent.
The selection of a gelling agent for specific plants is generally empirical. For unknown reasons,
tissues of some species grow more vigorously on one gelling agent than on another. Another major
consideration is the degree of hyperhydricity induced in a species by the different gelling agents. One
potential way to overcome this is to combine agar and either Gelrite or Phytagel in the medium.
E. Other supports
Mechanical supports such as filter paper folded into wicks and polyethylene rafts can be used with
liquid medium to ensure an adequate supply of oxygen. Many other materials have been used as well
including rock wool, cheesecloth, sand, and pieces of foam. Explants can be grown in rotating
cultures where the tissue is alternately bathed in liquid and exposed to air.
Media Formulations
Although the most used medium formulation is that of Murashige and Skoog (1962) (references are
in the Gamborg paper), many others have been developed. Murashige and Skoog (MS) medium was
developed for culture of tobacco and was formulated based on an analysis of the mineral
compounds present in the tobacco tissue itself. It has comparatively high salt levels, particularly of K
and N. Linsmaier-Skoog medium (Linsmaier and Skoog, 1965) is a version of MS medium with
modified organic constituents. White's medium (White, 1963), a low salt formulation, was developed
originally for the culture of tomato roots. Gamborg's B5 medium (Gamborg et al., 1968) was
developed for soybean callus culture and contains a much greater proportion of nitrate compared to
ammonium ions. The vitamins in this medium formulation are also often added to MS salts. Schenk
and Hildebrandt (1972) developed their medium for the culture of callus of both monocots and
dicots. Nitsch and Nitsch (1969) medium was developed for anther culture and contains lower salt
concentrations than MS medium, but not as low as in White's medium. Lloyd and McCown's
Woody Plant Medium (WPM) (Lloyd and McCown, 1981) has been used successfully for a great
many tree species. Knudson's medium has been used in orchard culture (Knudson 1946).
There are several important observations to make about these media:

All of them are fully defined (no complex organics).

A chelated iron source is preferred (Fe-EDTA).

MS and SH are high salt media.

MS and SH use both ammonium and nitrate ions as nitrogen sources.

SH contains a very high level of myo-inositol.

Sucrose is the carbon source.
Formulations of Common Plant Culture Media
Concentration in culture medium (mg/liter)
Constituent
MS
SH
B5
KNO3
1900
2500
2500
NH4NO3
1650
NH4H2PO4
300
(NH4)2SO4
134
MgSO4·7H2O
370
400
250
CaCl2·2H2O
440
200
150
KH2PO4
170
NaH2PO4·H2O
150
MnSO4·H2O
10.0
10.0
MnSO4·4H2O
22.3
KI
0.83
1.0
0.75
H3BO3
6.2
5.0
3.0
ZnSO4·7H2O
8.6
1.0
2.0
CuSO4·5H2O
0.025
0.2
0.025
Na2MoO4·2H2O
0.25
0.1
0.25
CoCl2·6H2O
0.025
0.1
0.025
FeSO4·7H2O
27.8
15.0
27.8
Na2EDTA
37.3
20.0
37.3
Nicotinic acid
0.5
5.0
1.0
Pyridoxine-HCl
0.5
0.5
1.0
Thiamine-HCl
0.1
5.0
10.0
myo-Inositol
100
1000
100
Glycine
2.0
Sucrose
30000
30000
20000
In complex media such as these, consisting of many components, there are a huge number of
permutations of substances and concentrations to test to compose an ideal medium for a particular
plant species and genotype. When working with a species new to you, the first place to start is in the
literature, finding out what other people have used for that species or one closely related. However,
there may be considerable differences in requirements for different cultivars. In practice, researchers
may test several basal media, but only try to optimize a few components, in particular, plant growth
regulators (PGRs).