NUTRITIONAL REQUIREMENTS CULTURE MEDIUM

                                                            NUTRITIONAL REQUIREMENTS FOR CULTURE MEDIUM

Requirements

1. Inorganic nutrients

2. Carbon / Energy source

3. Vitamins

4. Plant growth regulators (Auxins, cytokinins, gibberellins, abcicsins,ethylene, polyamines , organic acids)

5. Solidifying agent

6. Amino acids

7. Undefined supplements, and jasmonates

1. Inorganic nutrients

            A relatively small number of mineral salts are used as component of media for plant tissue culture. The inorganic salt formulations can vary in various reported media, however MS formulation is most widely used with or without modifications.

Major elements

• Calcium
• Iron
• Magnesium
• Nitrogen
• Phosphorus
• Potassium
• Sulfur

Minor elements

            Minor elements are required only in extremly small quantities. They are also called trace elements or micro-nutrients. Several trace elements are toxic to plants in excess amounts.

• Boron
• Chlorine
• Cobalt
• Copper
• Iodine
• Manganese
• Molybdenum
• Zinc

2. Carbon/Energy source

Carbohydrates

            Carbohydrates include such organic chemicals as sugars, starches, and cellulose and are made of C, H, and O. These elements are generously supplied as carbon dioxide (CO₂) in air and as water (H₂O).

i.                    sucrose

ii.                  D-Mannitol

iii.                D-Sorbitol

iv.                Hexitols

3. Vitamins

• Adenine
• D-Biotin
• Folic acid
• Inositol
• Nicotinic acid
• P-aminobenzoic acid
• Pyridoxine
• Riboflavin
• Thiamine
• L-Ascorbic acid
• α-tocopherol

4. Growth regulators

• auxin
• cytokinins
• gibberellins
• abscisic acid

5. Solidifying agent

            Commonly used solidifying agent is agar. Agar is mixture of polysaccharides derived from extracts of several species of red algae.Six to eight grams of agar per liter of medium is usually satisfactory, but gel strength will vary with the medium formula being used and the source and grade of agar.

6. Aminoacids

• L-Alanine
• L-Arginine
• L-Asparagine
• L-Serine

7. Undefined supplements

• coconut milk
• corn milk
• potato extract
• tomato juice
• yeast extract

Physical form of medium- solid and liquid media

Solidified Medium

Ø  Explants are easily seen and recovered

Ø  No Special aeration

Ø  Shoots grow in more orderly

Ø  Long term maintenance

Ø  Simple containers little space

Liquid medium

Ø  Recovery is difficult.

Ø  Usually shaken, rotated/aerated

Ø  Growth is disoriented

Advantages:

Ø  Callus break up and shed as cells to establish a fine suspension

Ø  Faster rate of multiplication

Ø  A greater surface of explants is in contact with medium.

Ø  Toxic metabolites will effectively be dispersed.

Disadvantages:

Ø  Seeds would not germinate; protocorms and plantlets became brown and dried. Seeds submerged will show vitrification.

Commonly used tissue culture media

MS (Murashige and Skoog, 1962) and LS (Linsmaier and Skoog, 1972) for plant regeneration of both monocots and dicots

B5 (Gamborg et al., 1969) developed for culture of soybean cell suspensions but also has been effectively used for methods of plant regeneration. B5 and its various derivatives have been valuable for cell and protoplast cultures.

SH: Schenk and Hildebrandt (1972) introduced this for culture of monocots and dicots, . Widely used especially for legumes.

WPM: Lloyd and McCown (1980,1981). This is post MS media.. WPM is increasingly used for propagation of Ornamental shrubs and trees in commercial labs.

N6 (Chu) was developed for cereal anther culture and used in the success in other cereal anther culture. In special cases , NN (Nitsch and Nitsch) was also used.

Environmental factors influencing plant tissue culture

Genotype and Explant:

Medium: (i)Nutrients, (ii)Growth regulators and (iii)Other additives

Culture Environment: (i) Temperature, (ii) Relative Humidity (RH) and (iii) Light