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The basic nutrient requirements of cultured plant cells are very similar to those of whole
plant. Culture media used for in vitro cultivation of plant cells are composed of following
basic components:
CONSTITUENTS SOURCES
Complex mixture of salts Microelements, Macroelements
Organic supplement Vitamins, Amino Acids
Carbon source Sucrose, Glucose, Fructose, Maltose
Gelling Agent Agar, Gelrite
Plant Growth Regulators Auxin, Cytokinins, Gibberellins, Abscisic Acid, Ethylene
Antibiotics Ampicillin, Tetracycline, Amphotericin B
NUTRIENTS: There are two types of nutrients, macronutrients which should be present in
amount greater than 0.5 mM in a media and micronutrients which are required in less
amount <0.5 mM.
Macro element: Nitrogen, Phosphorous, Potassium, Magnesium, Calcium and Sulphur are
These elements comprises at least 0.1% of dry weight of plants. The optimum concentration
of each nutrient for achieving maximum growth rate varies considerably among species. B5
and MS Media are rich, especially for K and N.
Microelement: These elements are required in
trace amounts for plant growth and development.
Manganese, Iodine, Copper, Cobalt, Boron,
Molybdenum, Iron and Zinc are regarded as
microelements, although other elements like
Aluminium and Nickel are frequently found in
some formulations.
Manganese, Copper, Zinc are enzyme cofactors.
Iron is a component of cytochrome and required
for electron transfer.
Generally, iron is added in the medium as Ferrous Sulphate, although Ferric citrate can also
be used. Iron-EDTA complex is usually added to keep it available at higher pH.
Uncomplexed Iron can precipitate out the medium as Ferric Oxide.
Carbon sources: The plant tissue culture media, though sucrose is very frequently used (at
the concentration 2-5%), there are many other carbohydrates that are also used, like lactose,
galactose, maltose and starch but they are less effective than either sucrose and glucose.
Glucose is more effective than fructose as it is used by the cells in the beginning.
Organic Substances: Vitamins are needed for metabolic processes in tissue culture. Since
tissue synthesize vitamins in low concentration therefore they have to be supplemented
from outside. Thiamine, nicotinic acid, pyrodoxin and calcium pantothenate are the vitamins
which are commonly supplemented.
Apart from this amino acid culture tissues are usually capable of synthesizing amino acid for
various metabolic processes but we need to supplement them for stimulating cell growth in
protoplast cultures and establishing cell lines. Glycine is most commonly used amino acid.
However, Arginine, Asparagine, aspartic acid, Alanine, glutamine and proline are also used
as source of amino acid.
Gelling Agents: Growth of cultured tissues is greatly
influenced by the hardness of culture medium. There
are number of gelling agent used like agar, agarose and
gellan gum. In plant tissue culture, agar has been used
as a solidifying agent since long. There are several
advantages of agar, over other gelling agent; like it get
mixed in water, gets easily melt at the temperature
range60 -100 C and forms a gel that is stable at all the
feasible incubation temperature. It is inert and thus do
not get reacted with media constituents and is not
Fig: TP 001A: MURASHIGE & SKOOG
digested by plant enzymes. MEDIUM
Plant Growth Promotor:
The growth regulators which are used in plant tissue culturing include: Auxins like Indole -3-acetic
acid (IAA), Indole-3-butyric acid (IBA), Napthalene Acetic acid (NAA), (Naphthoxy Acetic
Acid (NOA), 2,4-dicholorophenoxy acetic acid (2,4-D), to support cell division and callus
growth (especially 2,4-D), somatic embryo induction and rooting.Cytokinins like kinetin,
FAP(Furfurylamino purine),BAP (Benzylamino purine),TDZ (Thidiazuron) are employed to
promote cell division , regeneration of shoot, somatic embryo induction and proliferation
and growth of axillary buds. Abscisic Acid (ABA) promotes SE and shoot bud regeneration
in many species and markedly improves SE maturation.Among 20 gibberellins known, GA3
is exclusively used to promote shoot elongation and SE germination.Ehylene is associated
with controllong fruit ripening ams climatic fruits but is not widespread.
The concentrations used of various growth regulators are as follows: auxins (0.1-3 mg/l);cytokinins
(0.1-3mg/l);ABA (upto 0.2 mg/l); and GA3(0.1-1 mg/l).
Antibiotics:
Antibiotics are substances produced by certain microorganisms that suppress the growth of
other microorganisms and eventually destroy them. Their applications include:
A. Suppresses bacterial infections in plant cell tissue culture.
B. Suppresses mold and yeast infections in cell cultures.
C. Eliminates Agrobacterium species after the transformation of plant tissue.
These antibiotic can be divided into different classes on the basis of chemical structure and their
mechanism of action:
OSE MPLE
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