Professional Documents
Culture Documents
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.
I. Mineral elements
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.
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.
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.
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
D. Complex organics
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).
Constituent MS SH B5
NH4NO3 1650
NH4H2PO4 300
(NH4)2SO4 134
KH2PO4 170
NaH2PO4·H2O 150
MnSO4·4H2O 22.3
Glycine 2.0