Plant Tissue Culture Media

Plant Tissue Culture Media

Plant Tissue Culture refers to the technique of growing plant cells, tissues, organs, seeds or other plant parts in a sterile environment on a nutrient medium. Culture media used for in vitro cultivation of plant cells are composed of following basic components: Complex Mixture of Salts: Essential elements, or mineral ions Organic Supplement: vitamins and/or amino acids Carbon Source: usually sugar sucrose Gelling Agents Plant Growth Regulators Antibiotics

Complex Mixture of Salts

These include essential elements or mineral ions important for plant nutrition and their physiological function. The essential elements can further be divided into the following categories:

Macroelements (or macronutrients) Microelements (or micronutrients) Iron source   Macroelements :- These elements are required in large amounts for plant growth and development. Nitrogen, phosphorus, potassium, magnesium, calcium and sulphur (and carbon, which is added separately) are regarded as macroelements. These elements comprise at least 0.1% of the dry weight of plants.       Microelements :- 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.

Iron Source :- Iron is usually added in the medium as iron sulphate, although iron citrate can also be used. Ethylenediaminetetraacetic acid (EDTA) is usually used in conjunction with the iron sulphate. The EDTA complexes with the iron so as to allow the slow and continuous release of iron into the medium. Uncomplexed iron can precipitate out of the medium as ferric oxide.

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Organic Supplements

These include vitamins and amino acids. Two vitamins, i.e., thiamine (vitamin B1) and myoinositol (a vitamin B) are essential for the culture of plant cells in vitro. However, other vitamins are often added to for historical reasons. The most commonly used amino acid is glycine. However, arginine, asparagine, aspartic acid, alanine, glutamic acid, glutamine and proline are also used. Amino acids provide a source of reduced nitrogen and, like ammonium ions, uptake causes acidification of the medium. Casein hydrolysate can be used as a source of a mixture of amino acids.

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Carbon Source

The most commonly used carbon source is sucrose. It is readily assimilated and relatively stable. Other carbohydrates like glucose, maltose, galactose and sorbitol can also be used and may prove better than sucrose in specialized circumstances.

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Gelling Agents

Plant tissue culture media can be used in either liquid or ‘solid’ forms, depending on the type of culture being grown. Agar, produced from seaweed, is the most common type of gelling agent, and is ideal for routine applications. For more demanding applications, a range of purer gelling agents are available. Purified agar or agarose can be used, as can a variety of gellan gums.

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Plant Growth Regulators

Specific media manipulations can be used to direct the development of plant cells in culture due to plasticity and totipotency. Plant growth regulators are the critical media components in determining the developmental pathway of the plant cells. There are five main classes of plant growth regulator used in plant cell culture, namely: Auxins Cytokinins Gibberellins Abscisic Acid Ethylene

Auxins :- Auxins promote both cell division and cell growth. IAA (indole-3-acetic acid) is the most important naturally occurring auxin but its use in plant tissue culture media is limited because it is unstable to both heat and light. 2,4-Dichlorophenoxyacetic acid (2,4-D) is the most commonly used auxin and is extremely effective in most circumstances.   Cytokinins :- Cytokinins promote cell division. Of the naturally occurring cytokinins, only zeatin and 2iP (2-isopentyl adenine have some use in plant tissue culture media. The synthetic analogues, kinetin and BAP (benzylaminopurine), are used more frequently. Non-purine-based chemicals, such as substituted phenylureas, are also used as cytokinins in plant tissue culture media.   Auxins :- Auxins promote both cell division and cell growth. IAA (indole-3-acetic acid) is the most important naturally occurring auxin but its use in plant tissue culture media is limited because it is unstable to both heat and light. 2,4-Dichlorophenoxyacetic acid (2,4-D) is the most commonly used auxin and is extremely effective in most circumstances.

Cytokinins :- Cytokinins promote cell division. Of the naturally occurring cytokinins, only zeatin and 2iP (2-isopentyl adenine have some use in plant tissue culture media. The synthetic analogues, kinetin and BAP (benzylaminopurine), are used more frequently. Non-purine-based chemicals, such as substituted phenylureas, are also used as cytokinins in plant tissue culture media. Gibberellins :- Gibberellins are involved in regulating cell elongation, in determining plant height and fruit-set. Only a few of the gibberellins like GA3 are used in plant tissue culture media. Abscisic Acid :- It is used in plant tissue culture to promote distinct developmental pathways such as somatic embryogenesis. Abscisic acid (ABA) inhibits cell division. Ethylene :- Ethylene is associated with controlling fruit ripening in climacteric fruits, and its use in plant tissue culture is not widespread. Some plant cell cultures produce ethylene, which, if it builds up sufficiently, can inhibit the growth and development of the culture.

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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 and tissue culture. B. Suppresses mould and yeast infections in cell cultures. C. Eliminates Agrobacterium species after the transformation of plant tissue. These antibiotics can be divided into different classes on the basis of chemical structure and their mechanism of action: Inhibitors of Bacterial Cell Wall Synthesis   e.g. β-lactam antibiotics, Penicillins and Cephalosporins. Antibiotics that affect Cell Membrane permeability.     •  Antibacterial e.g. Colistin Sulphate, Polymixin B Sulphate, Gramicidin   •  Antifungal e.g. Amphotericin B, Nystatin, Pimaricin Bacteriostatic Inhibitors of Protein

Antibiotics that affect the function of 30 S or 50 S ribosomal subunits to cause a reversible inhibition of protein synthesis. e.g. Chloramphenicol, Chlortetracycline HCl, Clindamycin HCl, Doxycycline HCl, Erythromycin, Lincomycin HCl, Oxytetracycline HCl, Spectinomycin sulphate, Tetracycline HCl, Tylosin tartrate, Lincomycin HCl Bactericide Inhibitors of Protein Synthesis   Antibiotics that bind to the 30 S ribosomal subunit and alter protein synthesis which eventually leads to cell death. This group includes:   Aminoglycosides: Apramycin, Butirosine, Gentamicin, Kanamycin, Neomycin, Streptomycin,    Tobramycin. Inhibitors of Nucleic Acid Metabolism: e.g. Rifampicin, Mitomycin C and Nalidixic acid Antimetabolites: Antibiotics, which block specific metabolic steps that are essential to microorganisms    e.g.  Metronidazole, Miconazole, Nitrofurantoin, Trimethoprim and Sulphomethoxazole. Nucleic Acid Analogs, which inhibit enzymes essential for DNA synthesis. e.g. 5-Fluorouracil,    Mercaptopurine.

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Preparation of Plant Tissue Culture Medium

Measure approximately 90% of the required volume of the deionized-distilled water in a flask/container of double the size of the required volume. Add the dehydrated medium into the water and stir to dissolve the medium completely. Gentle heating of the solution may be required to bring powder into solution. Add desired heat stable supplements to the medium solution. Add additional deionized-distilled water to the medium solution to obtain the final required volume. Set the desired pH with NaOH or HCl. Dispense the medium into culture vessels. Sterilize the medium by autoclaving at 15 psi (121οC) for appropriate time period. Higher temperature   may result in poor cell growth. Add heat labile supplements after autoclaving.

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