3. plant tissuce culture media

Plant Tissue Culture Media
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PTC course for 4th stage Horticulture students. By: Dr. Rafail S. Toma

What is it?
• Is the place in which an explant grown in.
• It provides all the needs of plant growth
and development.
2

Functions of medium
• Provide water
• Provide mineral nutritional needs
• Provide vitamins
• Provide growth regulators
• Access to atmosphere for gas exchange
• Removal of plant metabolite wastes
3

Ingredients
• inorganic salts
• organic compounds
• gelling agents
4

Inorganic Salts
For healthy and vigorous growth, intact plants
need to take up from the soil:
• Relatively large amounts of some inorganic elements
(the so-called major plant nutrients): ions of nitrogen
(N), potassium (K), calcium (Ca), phosphorus (P),
magnesium (Mg) and sulphur (S); and,
• Small quantities of other elements (minor plant
nutrients or trace elements): iron (Fe), nickel (Ni),
chlorine (Cl), manganese (Mn), zinc (Zn), boron (B),
copper (Cu), and molybdenum (Mo).
5

Inorganic Salts
Medium salts are grouped into five distinct groups:
1. Nitrates (Ammonium nitrate, potassium nitrate)
2. Sulfates (Magnesium sulfate, Manganese sulfate, Zinc sulfate,
cupric sulfate)
3. Na, Fe EDTA (Ferrous sulfate, EDTA (Ethylene Diamine Tetra
Acetic acid Di-sodium Salt)
4. Halides (Calsium chloride, potassium iodide, cobalt chloride)
5. P, B, Mo (Potassium phosphate, boric acid, sodium molybdate)
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According to Epstein (1971), an element can
be considered to be essential for plant
growth if:
• 1. A plant fails to complete its life cycle without it;
• 2. Its action is specific and cannot be replaced
completely by any other element;
• 3. Its effect on the organism is direct, not indirect
on the environment;
• 4. It is a constituent of a molecule that is known to
be essential
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Nitrogen (N)
Promotes rapid vegetative growth
and gives plants healthy green
color.
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Phosphorus (P)
Stimulates early growth and root
formation, hastens maturity, promotes
seed production and makes plants hardy.
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Potassium (K)
Improves plant’s ability to resist disease
and cold, aids in the production of
carbohydrates.
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Calcium (Ca)
Aids in the movement of carbohydrates
in plants, essential to healthy cell walls
and root structure.
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Magnesium (Mg)
An ingredient of chlorophyll, aids in the
translocation of starch within the plant,
essential for formation of oils and fats.
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Sulfur (S)
Aids in the formation of oils and parts of
protein molecules.
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Boron (B)
Aids in the assimilation of calcium;
amount required is extremely small.
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Copper (Cu)
Promotes formation of Vitamin A,
excess is very toxic.
15

Manganese (Mn)
Serves as an activator for enzymes in
growth processes, assist iron in
chlorophyll formation, generally
required with zinc in foliar spraying
of citrus.
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Zinc (Zn)
An essential constituent of several
enzymes, controls synthesis of indoleacetic
acid - an important growth regulator.
The micronutrient most often needed by
western crops - trees, grapes, beans,
onions, tomatoes, cotton & rice.
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Molybdenum (Mo)
Required for N utilization, and
legumes cannot fix atmospheric N
symbiotically without Mo.
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Chlorine (Cl)
Required in photosynthetic reactions of
plants.
Deficiency is not seen in the field due to
its universal presence in nature.
19

Iron (Fe)
Essential for formation of
chlorophyll, releases energy from
sugars and starches.
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Kinds of nutrient media
There are many different formulations reported in the
literature with detailed descriptions.
• Murashige and Skoog (MS) (herbaceous plants)
• Linsmaier-Skoog media (herbaceous plants)
• The woody plant medium (WPM) (woody plants)
• Anderson (AND) medium (rhododendrons)
• Gamborg (B5) medium (widely used for cell and tissue
cultures).
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Organic Compounds
• Carbohydrates. Sucrose at 2 to 4 percent is
used for most cultures. Glucose, Fructose,
Maltose, and Starch have been used
occasionally.
• Vitamins. Thiamin (0.1 to 0.5 mg/l),
nicotinic acid (0.5 mg/l), pyridoxine (0.5
mg/l), Inositol at 100 mg/l, pantothenic
acid (0.1 mg/l) and biotin (0.1 mg/l).
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Organic Compounds
• Hormones and Growth regulators.
• Auxins. The natural auxin, IAA (1 to 50 mg/l). Synthetic
auxins, IBA (0.1 to 10 mg/l), 2, 4-D (0.05 to 0.5 mg/l) and
NAA (0.05 to 0.5 mg/l).
• Cytokinins. BA, kinetin, N6
–isopentyl-adenine (2iP), and
zeatin. (0.01 to 10 mg/l).
Thidiazuron and N-2-chloro-4-phenylurea (CPPU) have
cytokinin activity and are often used in combination with
traditional cytokinins (like BA).
Adenine sulfate also has cytokinin activity and is added to 40
to 120 mg/l.
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Gelling Agents and Supports
• Agar. Agar is obtained from certain species of red
algae and is commercially available in a powdered
form. Its value in culture systems is:
• ability to melt when heated
• change to a semisolid gel at room temperature
• relative biological inertness.
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Gelling Agents and Supports
• Agar Substitutes. Gelrite is a polysaccharide derived
from Pseudomonas bacteria. A ratio of 3: 1 Gelrite
to agar, Gelrite should be avoided for species
susceptible to hyperhydricity (vitrification).
25

Liquid Media
• Nutrient solutions without agar have an advantage for
some plants.
• Some types of support is required to keep explants and
cultures from sinking, or a shaker or rotating drum
must be used to provide aeration.
• Filter paper bridges may be inserted into the liquid to
support the culture with the paper acting as a wick.
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Liquid Media
• Plastic fabric supports and membrane rafts
• Cotton and vermiculite.
• Liquid media are useful for some plant species whose
explants exude toxic substances from the cut surfaces.
• The liquid medium can be exchanged without
reculturing.
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Composition
of the
premixed
MS nutrient
medium
Ingredients mgl-1
KNO3 .1900 00
NH4NO3 .1650 00
CaCl2.2H2O .332 2
MgSO4.7H2O .180 69
KH2PO4 .170 00
MnSO4.4H2O .22 30
H3BO3 .6 20
KI .0 83
Na2MoO4.2H2O .0 25
Sodium Phosphate monobasic 170
ZnSO4.7H2O .8 60
CuSO4.5H2O .0 025
Co(NO3)2.6H2O .0 025
FeSO4.7H2O .27 80
Na2-EDTA .37 30
Myo-Inositol .100 00

Preparation of the Medium
• Adding inorganic salts
• Adding organic compounds
• pH adjustment
• Adding gelling agents
• Dissolving by heating and stirring
• Dispensing
• Autoclaving
• Ready for culture after 24 hours
29

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