PP 508-MORPHOGENESIS, TISSUE CULTURE AND TRANSFORMATION

TISSUE CULTURE MEDIA ;

COMPOSITION AND TYPES

Femina k
2018-11-094
TISSUE CULTURE

 Tissue culture is the process of in-vitro culture of explant (tissues or

cells) in an artificial medium under aseptic conditions.
 This is typically facilitated via use of a liquid, semi-solid, or solid growth
medium
 Tissue culture Media

 Cells of most plant cells can be grown in culture media.

 The success of the plant tissue culture depends on the choice of
the nutrient medium.
 Media used in plant tissue culture contain nutritional components
which are essential for growth and development of cultured tissue
Composition of Media
must contain the following essential components to support invitro plant
growth.

1) Inorganic nutrients ( Micro and Macro nutrients)

2) Carbon sources
3) Organic supplements
4) Plant growth regulators
5) Gelling agent
Macro nutrients

 It is present in milli molar (mM) quantities

 Macro nutrients provide both anions and cations for the plant cells.

1) nitrogen (NO3 and NH4)

2) Phosphorus (PO4)
3) Potassium (K)
4) Sulphur (SO4)
5) Magnesium (Mg)
6) calcium (Ca)
Micro Nutrients

 Boron(B), Manganese(Mn), Zinc(Zn), Molybdenum(Mo), Copper(Cu),

Cobalt(Co), Iron (Fe)
 Used in less amount less than 30ppm
 Concentration is always in µM.
Carbon sources

 Cells and tissue requires exogenous supply of carbohydrates to replace

the carbon which the plant normally fixes
 The preferred carbohydrate in plant cell culture media is sucrose (2-5 %)
 During autoclaving sucrose will hydrolyses in to fructose and glucose
 Glucose, fructose, lactose and maltose may be substituted in some cases
 Concentration is 20-30 gm/l.
Organic nutrients
Vitamins (0.1-10 mg/ml)
 Essential for many biochemical reaction.
 Cultured cells require an exogenous supply of different vitamins for
optimum growth.
 Most usable vitamins are Thiamine, Pyridoxine, biotin, folic acid,
riboflavin, nicotinic acid and Vitamin B Complex.
 Thiamin is the one vitamin that is basically required by all cells for
growth.
Amino Acids

 The use of amino acids is particularly important for establishing cell cultures
and protoplast cultures.
 Amino acids provide plant cells with an immediately available source of
nitrogen, which generally can be taken up by the cells more rapidly than
inorganic nitrogen.
 The most common sources of organic nitrogen used in culture media are
amino acid mixtures
 Casein hydrolysate, L-glutamine, L-asparagine, L-glycine and adenine.
Natural supplements

 Coconut milk
 Fruit materials
 Potato extract
 Extracts of malts, yeast
 Orange juice
 Tomato juice
 Banana pulp
 Fish emulsion
 Peptone
 Plant growth regulators

 Auxins
 Essential for cell division, cell elongation, cell differentiation, organogenesis
and embryogenesis, callus formation
 Natural form auxins are IAA, IBA, PAA
 Synthetic form of auxins are NAA, 2, 4-D.
 Cytokinins
 Cytokinins promote cell division, shoot proliferation and influence the cell
cycle, Embryogenesis and inhibit roof formation.
 Natural forms are BAP ( Benzyl amino purine), zeatin and kinetin.
 Gibberellins
 It promotes stem elongation, bulb corm formation and embryo maturation
but can inhibit callus growth and root induction.
 GA3 is most common gibberellins.

 Abscisic acid
 It inhibits shoot growth but most often requires for normal growth and
development of somatic embryos
 It is thermostable but light sensitive
Gelling Agent-Agar
 Agar has long been used to solidify media for plant tissue culture.
 When agar is mixed with water, it forms a gel that melts at approximately
60°-100° C and solidifies at approximately 45°C; thus, agar gels are stable at
all feasible incubation temperatures.
 Agar gels do not react with media constituents and are not digested by plant
enzymes.
 The agar concentrations commonly used in plant cell culture media range
between 0.5 and 1.0%
 Gelrite- Synthetic and should be used at 1.25-2.5 g/liter, resulting in a clear
gel which aids in detecting contamination.
 Alginate can also use as an alternative
PH of tissue culture media

 PH is adjusted between 5 & 5.8 before gelling and sterilization with the
help of dilute NaOH, KOH or HCL.
 below 5 will not gel properly.
 above 6 may be too hard.
Major Types of Media

 MS medium:
Murashige and Skoog (MS) originally formulated a medium to induce
organogenesis, and regeneration of plants in cultured tissues. It has a high
concentration of nitrate, ammonium and potassium
 White’s medium
This is one of the earliest plant tissue culture media developed for root
culture. Used in callus culture and cell suspension culture. Nitrate
concentration is 19% less than MS medium.
 B5 medium
Developed by Gamborg. B5 medium was originally designed for cell suspension
and callus cultures. At present with certain modifications, this medium is used for
protoplast culture.

 N6 medium
Chu formulated this medium and it is used for cereal anther culture, besides other
tissue cultures.

 Nitsch’s medium
This medium was developed by Nitsch and frequently used for anther cultures.

Among the media referred above, MS medium is most frequently used in plant
tissue culture work due to its success with several plant species and culture
systems.
 Synthetic and natural media

 When a medium is composed of chemically defined components, it is referred

to as a synthetic medium.
 On the other hand, if a medium contains chemically undefined compounds
(e.g., vegetable extract, fruit juice, plant extract), it is regarded as a natural
medium.
 Synthetic media have almost replaced the natural media for tissue culture.
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