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Tissue Culture

Plasticity and totipotency

Two concepts, plasticity and totipotency, are central to understanding plant cell culture and regeneration. Plants, due to their sessile nature and long life span, have developed a greater ability to endure extreme conditions and predation than have animals. Many of the processes involved in plant growth and development adapt to environmental conditions. This plasticity allows plants to alter their metabolism, growth, and development to best suit their environment. Particularly important aspects of this adaptation, as far as plant tissue culture and regeneration are concerned, are the abilities to initiate cell division from almost any tissue of the plant and to regenerate lost organs or undergo different developmental pathways in response to particular stimuli. When plant cells and tissues are cultured in vitro they generally exhibit a very high degree of plasticity, which allows one type of tissue or organ to be initiated from another type. In this way, whole plants can be subsequently regenerated.

This regeneration of whole organisms depends upon the concept that all plant cells can, given the correct stimuli, express the total genetic potential of the parent plant. This maintenance of genetic potential is called totipotency. Plant cell culture and regeneration do, in fact, provide the most compelling evidence for totipotency. In practical terms though, identifying the culture conditions and stimuli required to manifest this totipotency can be extremely difficult and it is still a largely empirical process.

Laboratory Requirements for Tissue Culture

General Organization

Localize each portion of the tissue culture procedure in a specified place in the laboratory. An assembly-line arrangement of work areas (such as, media preparation, glassware washing, sterilization, microscopy, and aseptic transfers) facilitates all operations and enhances cleanliness. Media (tissue culture and nutrient agar) are available from Carolina Biological Supply Co., Burlington, NC. Laminar flow hoods are available from several suppliers.

Glassware

Use glassware that has only been used for tissue culture and not other experiments. Toxic metal ions absorbed on glassware can be especially troublesome. Wash glassware with laboratory detergent, then rinse several times with tap water and, finally, rinse with purified water.

High-purity Water

Use only high-purity water in tissue culture procedures. Double glass distilled water or deionized water from an ion-exchanger are acceptable. Water should not be stored, but used immediately. Regular maintenance and monitoring of water purification equipment are necessary. Purified water for tissue culture can also be purchased.

Plant Material

Plants used in tissue culture need to be healthy and actively growing. Stressed plants, particularly water-stressed plants, usually do not grow as tissue cultures. Insect and disease-free greenhouse plants are rendered aseptic more readily, so contamination rate is lower when these plants are used in tissue culture procedures. Seeds that can be easily surface sterilized usually produce contamination-free plants that can be grown under clean greenhouse conditions for later experimental use.

Aseptic Technique

The essence of aseptic technique is the exclusion of invading microorganisms during experimental procedures. If sterile tissues are available, then the exclusion of microorganisms is accomplished by using sterile instruments and culture media concurrently with standard bacteriological transfer procedures to avoid extraneous contamination.

Media and apparatus are rendered sterile by autoclaving at 15 lbs/inch2 (121°C) for 15 minutes. The use of disposable sterile plastic ware reduces the need for some autoclaving. Alternative sterilization techniques such as filter sterilization must be employed for heat-labile substances like cytokinins.

Aseptic transfers can be made on the laboratory bench top by using standard bacteriological techniques (i.e., flaming instruments prior to use and flaming the opening of receiving vessels prior to transfer). Aseptic transfers are more easily performed in a transfer chamber such as a laminar flow hood, which is also preferably equipped with a bunsen burner.

The culture environment

When cultured in vitro, all the needs of the plant cells, both chemical (see Table 2.1) and physical, have to met by the culture vessel, the growth medium, and the external environment (light, temperature, etc.). The growth medium has to supply all the essential mineral ions required for growth and development. In many cases (as the biosynthetic capability of cells cultured in vitro may not replicate that of the parent plant), it must also supply additional organic supplements such as amino acids and vitamins. Many plant cell cultures, as they are not photosynthetic, also require the addition of a fixed carbon source in the form of a sugar (most often sucrose). One other vital component that must also be supplied is water, the principal biological solvent. Physical factors, such as temperature, pH, the gaseous environment, light (quality and duration), and osmotic pressure, also have to be maintained within acceptable limits.

Some elements important for plant nutrition and their physiological function, supplied by the culture medium to support the growth of healthy cultures in vitro,

Element                                   Function

Nitrogen                                  Component of proteins, nucleic acids, and some coenzyme

Potassium                               Regulates osmotic potential; principal inorganic cation

Calcium                                   Cell-wall synthesis, membrane function, cell signaling

Magnesium                            Enzyme cofactor, component of chlorophyll

Phosphorus                            Component of nucleic acids; energy transfer

Sulphur                                   Component of some amino acids (methionine, cysteine) and

Chlorine                                  Required for photosynthesis

Iron                                          Electron transfer as a component of cytochromes

Manganese                             Enzyme cofactor

Cobalt                                      Component of some vitamins

Copper                                     Enzyme cofactor; electron-transfer reactions

Zinc                                           Enzyme cofactor; chlorophyll biosynthesis

Molybdenum                          Enzyme cofactor; component of nitrate reductase

Plant cell culture media

Culture media used for the cultivation of plant cells in vitro are composed of three basic components:

1 essential elements, or mineral ions, supplied as a complex mixture of salts;

2 an organic supplement supplying vitamins and/or amino acids; and

3 a source of fixed carbon; usually supplied as the sugar sucrose.

For practical purposes, the essential elements are further divided into the following

categories:

1 macroelements (or macronutrients);

2 microelements (or micronutrients); and

3 an iron source.

Media components

It is useful to briefly consider some of the individual components of the stock solutions.

Macroelements

As is implied by the name, the stock solution supplies macroelements required in large amounts for plant growth and development. Nitrogen, phosphorus, potassium, magnesium, calcium, and sulphur (and carbon, which is added separately) are usually regarded as macroelements. These elements usually comprise at least 0.1% of the dry weight of plants.

Nitrogen is most commonly supplied as a mixture of nitrate ions (from KNO3) and ammonium ions (from NH4NO3). Theoretically, there is an advantage in supplying nitrogen in the form of ammonium ions, as nitrogen must be in the reduced form to be incorporated into macromolecules. Nitrate ions therefore need to be reduced before incorporation. However, at high concentrations, ammonium ions can be toxic to plant cell cultures and uptake of ammonium ions from the medium causes acidification of the medium. For ammonium ions to be used as the sole nitrogen source, the medium needs to be buffered. High concentrations of ammonium ions can also cause culture problems by increasing the frequency of vitrification (the culture appears pale and ‘glassy’ and is usually unsuitable for further culture). Using a mixture of nitrate and ammonium ions has the advantage of weakly buffering the medium as the uptake of nitrate ions causes OH− ions to be excreted.

Phosphorus is usually supplied as the phosphate ion of ammonium, sodium, or potassium salts. High concentrations of phosphate can lead to the precipitation of medium elements as insoluble phosphates.

Microelements

Microelements are required in trace amounts for plant growth and development, and have many and diverse roles. Manganese, iodine, copper, cobalt, boron, molybdenum, iron, and zinc usually comprise the microelements, although other elements such as nickel and aluminium are found frequently in some formulations.

Organic supplements

Only two vitamins, thiamine (vitamin B1) and myoinositol (considered a B vitamin), are considered essential for the culture of plant cells in vitro. However, other vitamins are often added to plant cell culture media for historical reasons. Amino acids are also commonly included in the organic supplement. The most frequently used is glycine (arginine, asparagine, aspartic acid, alanine, glutamic acid, glutamine, and proline are also used), but in many cases its inclusion is not essential. 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 relatively cheap source of a mix of amino acids.

Carbon source

Sucrose is cheap, easily available, readily assimilated, and relatively stable, and is therefore the most commonly used carbon source. Other carbohydrates (such as glucose, maltose, galactose, and sorbitol) can also be used (see Chapter 3), and in specialized circumstances may prove superior to sucrose.

Gelling agents

Media for plant cell culture in vitro can be used in either liquid or ‘solid’ forms, depending on the type of culture being grown. For any culture types that require the plant cells or tissues to be grown on the surface of the medium, it must be solidified (more correctly termed gelled). Agar, produced from seaweed, is the most common type of gelling agent, and is ideal for routine applications. However, because it is a natural product, the agar quality can vary from supplier to supplier and from batch to batch. For more demanding applications (see, for instance, the section on microspore culture below and Chapter 3), a range of purer (and in some cases, considerably more expensive) gelling agents are available. Purified agar or agarose can be used, as can a variety of gellan gums.

Plant growth regulators

We have already briefly considered the concepts of plasticity and totipotency. The essential point as far as plant cell culture is concerned is that, due to this plasticity and totipotency, specific media manipulations can be used to direct the development of plant cells in culture.

Classes of plant growth regulator

There are five main classes of plant growth regulator used in plant cell culture, namely:

(1) Auxins;

(2) Cytokinins;

(3) Gibberellins;

(4) abscisic acid;

(5) Ethylene.