Nutrient Media for Plant Tissue Cultures

Nutrient Media for Plant Tissue Cultures

One of the first decisions that must be made when developing a tissue culture system is what medium to use. 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. Not only did they find that the ions themselves were important, but the forms in which the ions were supplied were critical as well.

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. Most inorganic nitrogen is converted to amino acids and then to proteins. 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. In devising media, both the total amount of nitrogen as well as the relative amounts of NO3- and NH4+ are important. There are usually lower levels of NH4+ than NO3- in medium; nitrate is usually added at concentrations between 25 and 20 mM and ammonium at concentrations between 2 and 20 mM. For example, the amount of NH4+ in MS medium is less than half that of NO3- and in other media the NH4+ concentration is lower still. Cultures of some species can proliferate on medium containing nitrates alone, and some can grow on a medium with ammonium as the sole inorganic nitrogen source if one or more of the TCA cycle acids (citrate, succinate, malate) are included in the medium at concentrations of about 10 mM. In poorly buffered media, the use of both nitrogen forms helps maintain pH. Also, many plant species appear to respond best if they are given both forms, although the reason for this is not known.

Nitrogen may also be added to medium in an organic form, as amino acids such as proline or glutamine, hydrolysates (such as casein hydrolysate), or, as above, as organic acids. Organic nitrogen is already reduced, i.e. in the form in which most nitrogen exists in the plant, and so may be taken up more readily than inorganic nitrogen. The organic forms are often added to media that do not contain ammonium. However, almost always, some inorganic nitrogen is present.

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.

B. Micronutrients

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. The major problem in supplying iron in vitro is that it forms insoluble compounds in alkaline pH, a problem that is particularly evident in liquid culture, where it may be seen as a precipitate. The use of chelating agents, which bind metal ions, makes iron more stable and available to plant tissues over wider pH ranges. Although there are several of these, the sodium or potassium form of ethylenediaminetetraacetic acid (EDTA) is most often used because it is not as toxic as other chelating agents and it enables iron to be available to cultures over a wider pH range than other agents. Fe-EDTA may be purchased as a salt or prepared from ferric sulfate and EDTA.

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.

A. Sugars

Most plant tissue cultures are not highly autotrophic, that is, capable of fixing carbon through photosynthesis, due to limitations in culture of CO2 availability, among other factors. Therefore, sugar is added to the medium as an energy source. Sucrose is the most common sugar added, although glucose, fructose, and sorbitol are also used in certain instances. Sucrose is the sugar form most commonly transported in plants; it is broken down into glucose and fructose during metabolism. It is also partially hydrolyzed into glucose and fructose during autoclaving. The concentration of sugars in nutrient media generally ranges from 20 to 40 g/l.

Sugars also act as an osmoticum in the medium. Osmotic potential can have an important effect on in vitro response. Nutrient salts contribute from 20% to 50% of the osmotic potential of media, with sucrose making up the rest. When sucrose is hydrolyzed, as during autoclaving, its contribution to the osmotic potential is further increased.

B. Vitamins

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. Other vitamins such as biotin, folic acid, ascorbic acid (vitamin C), and vitamin E (tocopherol) are sometimes added to media formulations. Vitamin concentrations are generally very low.

C. Myo-inositol

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. Complex organics

These are undefined supplements such as coconut milk, coconut water, yeast extract, fruit juices and fruit pulps. They may supply amino acids, vitamins, plant growth regulators, and/or secondary plant metabolites. Complex organics were frequently used early in the history of plant tissue culture, when growth requirements were less defined. Now they are used only when no combination of defined components supports growth. Their disadvantages are that the important compounds in them are not known, and may vary greatly from batch to batch. Only protein hydrolysates and coconut milk (at 5-20% v/v) are used much today.

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. However, activated charcoal adsorbs not only toxic compounds, but also growth regulators and other compounds that are added to the medium. Activated charcoal is usually acid-washed prior to addition to the culture medium at a concentration of 0.5-3.0 %.

IV. Solidifying Agents

Solidifying agents are used to create semi-solid or solid media wherein plant cultures are not submerged in the medium. Liquid medium can be used for many plants but it must usually be agitated to provide sufficient oxygen to the tissue.

A. Agar

Agar is the most commonly used gelling agent. Marine red algae contain the structural polysaccharide agar, which consists of 2 components, agarose and agaropectin. Agarose is composed of alternating D-galactose and 3,6-anhydro-L-galactose with side chains of 6-methyl-D-galactose residues. Agaropectin is like agarose but additionally contains sulfate ester side chains and D-glucuronic acid. The tertiary structure of agarose is a double helix with a so-called threefold screw axis. The central cavity of this double helix can accommodate H2O molecules. Agarose and agaropectin readily form gels that contain high amounts of H2O (up to 99.5%). 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. All agar contains impurities, such as inorganic salts, organic compounds, phenolics, and long chain fatty acids; amounts and types vary depending on the manufacturer. These compounds usually do not interfere with culture response. If necessary, agar can be washed to remove inhibitory impurities. Agar does not gel well under acidic conditions (pH <4.5). The inclusion of activated charcoal in media may also inhibit gelling of agar. 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. The concentration of agar may be critical to plant response in culture. Medium that is too soft may produce hyperhydric (abnormal, glassy-looking) tissue while medium that is too hard may cause reduced growth.

B. Agarose

When greater purity is needed, agarose may be used. Agarose is extracted from agar leaving behind agaropectin and its sulfate groups. Because of the additional purification, agarose is considerably more expensive than agar. Agarose also has higher gel strength than agar and thus less is required for solidification of media. Agarose is used in situations where the impurities of agar are a major

Disadvantage, such as in protoplast culture.

C. Gelrite

Gelrite™ consists of a polysaccharide produced by the bacterium Pseudomonas elodea. Medium solidified with Gelrite has the advantage of being clear, which agar-solidified medium is not. Consequently contamination is more easily detected at an early stage. Impurities in Gelrite contain inorganic ions, but no organic compounds. Gelrite requires more stirring than agar when being added to media. Unlike agar, Gelrite cannot be reheated and gelled successfully. One limitation of Gelrite is that the concentration of divalent cations such as calcium and magnesium ions must be within the range of 4-8 mM/liter. Concentrations of these two ions either less than or greater than this range result in the media not gelling. Gelrite™ may also produce hyperhydric plants when used at low concentrations.

D. Phytagel

Phytagel™ is an agar substitute produced from a bacterial substrate composed of glucuronic acid, rhamnose and glucose. It produces a clear, colorless, high-strength gel, which aids in detection of microbial contamination. Phytagel provides an economical alternative to agar as a gelling agent. It is used at a concentration of 1.5-2.5 g/L. To prevent clumping, Phytagel should be added to rapidly stirring culture medium which is at room temperature. Hyperhydricity may also be a problem with this gelling agent.