Nutrient Media for Plant Tissue Cultures
Authors: Prashant Raghunath Shingote, and Dipti Raghunath Dhumale
Nutrient media for plant tissue culture are designed to allow plant tissues to be maintained in a totally artificial environment. Many different tissue culture media have been developed, but only a few have found wide-spread use, e.g. MS (Murashige and Skoog, 1962). SH (Shenck and Hildebrandt), and Gamborg's B5. One of the most successful media, devised by Murashige and Skoog (Murashige and Skoog, 1962) was formulated by analyzing the inorganic components in tobacco plants and then adding them to medium in amounts similar to those found in the plants.
In addition to mineral elements, the macro- and micronutrients that are similar to what is found in fertilizers, nutrient media also contain organic compounds such as vitamins, plant growth regulators, and a carbon source.
I. Mineral elements
A. Macroelements consist of N, K, P, Ca, Mg, and S.
1. Nitrogen (N) - Nitrogen is essential for plant growth. Nitrogen is typically added to plant nutrient media as the nitrate ion (NO3-, oxidized) and/or the ammonium ion (NH4+, reduced), which are added as inorganic salts. Inorganic nitrogen generally ranges from 25-60 mM in nutrient media.
2. Potassium (K) - Potassium is the major ion in plants with a positive charge, balancing negative ions. Although the amount of potassium required varies widely among different species, in media potassium concentration is generally correlated with that of nitrate and ranges between 20-30 mM.
3. Phosphorous (P) - Phosphorus is an integral part of nucleic acids and other structural compounds. It is added to culture medium as phosphate (PO4-) in sodium or potassium hydrogen phosphates in concentrations ranging from 1-3 mM.
4. Calcium (Ca) - Calcium is a co-factor of many enzymes and is particularly important in cell wall synthesis. It is supplied mostly as calcium chloride or calcium nitrate, concentrations ranging between 1 and 3 mM. In plant cultures, calcium deficiency may result in shoot tip necrosis.
5. Magnesium (Mg) - Magnesium is critical for the functioning of enzymes, is an integral component of the chlorophyll molecule, and is a cation that balances negative ions. It is usually added as magnesium sulfate in concentrations similar to that of calcium.
6. Sulfur (S) - Sulfur is a part of several amino acids and has an important function in protein structure. It is supplied as the SO4- ion, generally with magnesium as the cation, in concentrations ranging from 1-3 mM.
Micronutrients used in plant tissue culture are Fe, Mn, Zn, B, Cu and Mo, Co, and I.
1. Iron (Fe) - Iron is necessary for chlorophyll synthesis and functions in many oxidation/reduction reactions. It is generally present in media at approximately 1 mM.
2. Manganese (Mn) - Manganese is required for enzyme reactions, particularly in respiratory and photosynthetic processes and is usually added as manganese sulfate in concentrations of 5-30 mM.
3. Zinc (Zn) - Zinc is also required in many enzyme activities and is added to medium in concentrations similar to that of manganese. The most common form in which zinc is added is as the sulfate salt.
4. Boron (B) - Boron is an essential element involved in lignin biosynthesis and metabolism of phenolic acids and is supplied as boric acid in culture medium (25-100 mM). Boron deficiency results in the death of shoot tip meristems.
5. Copper (Cu) - Copper is critical in many enzyme reactions, including the cytochrome oxidase system. It is added to culture medium (as cupric sulfate) in very low concentrations (0.1 mM), because high amounts can be toxic.
6. Molybdenum (Mo) - Molybdenum functions in the transformation of nitrate to ammonium. It is added as sodium molybdate in low concentrations (1 mM) in culture medium.
7. Cobalt (Co) - Cobalt is not considered to be an essential mineral by plant physiologists, but is included in many of the most widely used media formulations. Cobalt is supplied in concentrations similar to that of copper, again because it may be toxic at higher concentrations.
8. Iodine (I) - Iodine is not considered to be an essential element, but it is often added to plant culture media (5 mM) because it has been found to improve growth of roots and callus in vitro.
9. Other elements - Nickel (Ni), aluminum (Al), and silicon (Si), are added to a few media formulations. These elements have not been found to be necessary for most plant species in vitro.
II. Organic Compounds
Organic compounds are also added to plant culture medium. Some of these compounds, such as sugars, are absolutely needed for growth, while others, such as vitamins, undefined compounds, and organic acids, may not be essential but may enhance growth.
Most plant tissue cultures are not highly autotrophic. Therefore, sugar is added to the medium as an energy source. Sucrose is the most common sugar added.Sucrose is the sugar form most commonly transported in plants. The concentration of sugars in nutrient media generally ranges from 20 to 40 g/l.Sugars also act as an osmoticum in the medium.
Only thiamine (vitamin B1), which is required for carbohydrate metabolism and the biosynthesis of some amino acids, has been shown to be essential for most plant cultures. Nicotinic acid (niacin) and pyridoxine (B6) are also commonly added to Murashige and Skoog medium and some other media.
Myo-inositol, a sugar alcohol, is added to most plant culture media. Although not essential for culture viability, it can significantly improve in vitro response, especially in monocots. Although myo-inositol is not essential for growth of many plant species, its effect on growth is significant.
D. Plant Hormones
Auxins, abscisic acid, cytokinins, ethylene, and gibberellins are naturally occurring plant hormones. Auxins, cytokinins, and auxin-cytokinin interactions are most important for regulating growth and organized development in plant tissue and organ cultures. Abscisic acid, Ethylene,
Gibberellins and other hormone-like compounds have regulatory roles to ensure organogenesis or cell proliferation. Rather, these hormones are being synthesized in the tissues and are playing an active, but hidden role in growth and development. The added auxins and cytokinins will interact with these other endogenous plant hormones.
Commonly used synthetic auxins in tissue culture are 2,4-dichlorophenoxyacetic acid (2,4-D; often used for callus induction and suspension cultures). Other are NAA, IAA, IBA which used for rooting purpose. Whereas BAP, Zeatine, kinetine and TDZ are widely used cytokinine.
E. Organic supplements:
Organic suppliments like, Glutamine, Glycine, Cystine, casein hydrilysate, Proline are used in nutrient media for plant tissue cultura media.
III. Activated Charcoal
Activated charcoal is sometimes added to media in order to adsorb toxic compounds released by plant tissues, particularly oxidized phenolics. It may be especially useful in rooting medium. Activated charcoal is usually acid-washed prior to addition to the culture medium at a concentration of 0.5-3.0 %.
IV. Agar Solidifying Agents Agar is the most commonly used gelling agent. Marine red algae contain the structural polysaccharide agar, when agar is mixed with liquid, it forms a gel that melts at about 100° C and solidifies at about 45° C. Other benefits are that agar does not react with any components of the medium and it is not digested by enzymes from the plant tissue. Agar does not gel well under acidic conditions (pH <4.5). The agar concentrations commonly used in plant culture media range between 0.5% and 1%; these concentrations yield a firm gel at typical media pHs.
About Author / Additional Info:
I have completed my graduation and post graduation in Agricultural biotechnology. From last 4-5 years I have been working on different aspects of plant molecular genetics and functional genomics. I have published nearby 10 publication maximum of which are in peer reviewed journals having impact factors.