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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 µ 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 µ 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 µ 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 µ 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 µ 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 µ 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-D-galactose 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).