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Friday, September 28, 2012

Basics on Plant and Animal Tissue Culture

Animal Cell Culture
A wider range of ingredients needed to support survival and proliferation or differentiation. Invitro animal cell cultivation requires a complex combination of nutrients,considering glucose and glutamine as main carbon, energy and nitrogen sources.Mineral salts, amino acids and vitamins are also required; other essential nutrients, like growth factors, hormones, and receptor and transport proteins are required in small quantities as well. The pH was maintained at 7.4 and often it includes pH indicator phenol red (red at 7.4, yellow at 6.5, purple at 7.8). A typical media may or may not comprise of serum. The latter is called a serum-free media. Some of the common sources of serum can be fetal bovine serum, equine serum, calf serum etc. both the types of media have their own set of advantages and disadvantages.

The culture media is prepared in such a way that it provides
  1. The optimum conditions of factors like pH, osmotic pressure, etc.
  2. It should contain chemical constituents which the cells or tissues are incapable of synthesizing. Generally the media is the mixture of inorganic salts and other nutrients capable of sustaining cells in culture such as amino acids, fatty acids, sugars, ions,trace elements, vitamins, cofactors, and ions. Glucose is added as energy source - its concentration varying depending  on the requirement. Phenol Red is added as a pH indicator of the medium.
Basic Components in the Culture Media

Most animal cell culture media are generally having following 10 basic components and
they are as follows:

1. Energy sources: Glucose, Fructose, Amino acids
2. Nitrogen sources: Amino acids
3. Vitamins: Generally water soluble vitamins B & C
4. Inorganic salts: Na+, K+, Ca2+, Mg2+
5. Fat and Fat soluble components: Fatty acids, cholesterol.
6. Nucleic acid precursors
7. Antibiotics
8. Growth factors and hormones
9. pH and buffering systems
10. Oxygen and CO2 concentration.

Animal cell culture media vary in their complexity but most contain:

Amino acids 0.1-0.2 mM
Vitamins ca. 1 μM
Salts NaCl 150 mM
KCl 4-6 mM
CaCl 1 mM
Glucose 5-10 mM

Culture Media
A cell culture medium is composed of a number of ingredients and these ingredients vary from one culture medium to another. The nutrient media used for culture of animal cells and tissues must be able to support their survival as well as growth, i.e., must provide nutritional, hormonal factors.

The various types of media used for tissue culture may be grouped into two broad categories:

1. Natural media
2. Artificial media.

The choice of medium depends mainly on the type of cells to be cultured (normal,immortalized or transformed), and the objective of culture (growth, survival,differentiation, production of desired proteins). Non transformed or normal cells (finite life span) and primary cultures from healthy tissues require defined quantities of proteins, growth factors and hormones even in the best media developed so far. But immortalized cells (spontaneously or by transfection with viral sequences) produce most of these factors, but may still need some of the growth factors present in the serum.

In contrast, transformed cells (autonomous growth control and malignant properties) synthesize their own growth factors; in fact, addition of growth factors may even be detrimental in such cases. Buteven these cultures may require factors like insulin,transferrin, silenite, lipids, etc.

Natural Media

These media consist solely of naturally occurring biological fluids and are of the following three types:

1. Coagula or clots
2. Biological fluids
3. Tissue extracts

The natural biological fluids are generally used for organ culture. For cell cultures, artificial media with or without serum are used.

The most commonly used clots are plasma clots, which have been in use for a long time. Plasma is now commercially available either in liquid or lyophilized state. It may also be prepared in the laboratory, usually from the blood of male fowl, but blood clotting must be avoided during the preparation.

Biological Fluids
Of the various biological fluids used as culture medium, serum is the most widely used.
Serum is one of the very important components of animal cell culture which is the source of various amino acids, hormones, lipids, vitamins, polyamines, and salts containing ions such as calcium, ferrous, ferric, potassium etc. It also contains the growth factors which promotes cell proliferation, cell attachment and adhesion factors. Serum may be obtained from adult human blood, placental cord blood, horse blood or
calf blood (foetal calf serum, newborn calf serum, and calf serum); of these foetal calf serum is the most commonly used. Serum is the liquid exuded from coagulating blood.Different preparations of serum differ in their properties; they have to be tested for sterility and toxicity before use.

Tissue Extracts
Tissue or organ extracts and/or hydrolysates (e.g., bovine pituitary extract (BPE), bovine brain extract,chick embryo extract and bovine embryo extract), and animal-derived lipids and fatty acids, peptones, Excyte, sterols (e.g., cholesterol) and lipoproteins (e.g., high-density and low-density lipoproteins(HDLs and LDLs, respectively) are used in culturing of animal cells. Tissue extracts for example, Embryo extracts—Other biological fluids used as natural media include amniotic fluids,ascetic and pleural fluids, aqueous humour (from eye), serum ultra filtrate, insect haemolymph etc. Chick embryo extract is the most commonly used tissue extract, but bovine embryo extract is also used. Other tissue extracts that have been used are spleen, liver, bone marrow, etc. extracts. Tissue extracts can often be substituted by a mixture of amino acids and certain other organic compounds.

Artificial Media
Different artificial media have been devised to serve one of the following purposes:
1. Immediate survival (a balanced salt solution, with specified pH and osmotic pressure is adequate),
2. Prolonged survival (a balanced salt solution supplemented with serum, or with suitable formulation of organic compounds),
3. Indefinite growth
4. Specialized functions.

The various artificial media developed for cell cultures may be grouped into the following four classes:
(i) Serum containing media
(ii) Serum free media
(iii) Chemically defined media
(iv) Protein free media.

Liquid yellowish, clear content left over after fibrin and cells are removed from the blood is known as serum. Calf (bovine), foetal bovine, or horse are used, in some cases human. Fetal bovine serum (FBS)(10-20% v/v) is the most commonly applied supplement in animal cell culture media. Normal growth media often contain 2-10% of serum.  These supplements provide carriers or chelators for labile or water-insoluble nutrients; bind and neutralize toxic moieties; provide hormones and growth factors,protease inhibitors and essential, often unidentified or undefined low molecular weight nutrients; and  protect cells from physical stress and damage. Thus, serum and/or animal extracts are commonly used as relatively low-cost supplements to provide an optimal culture medium for the cultivation of animal cells. The role for all constituents (more than 200) is not clear proteins, peptides, special factors released during platelet aggregation e.g., PDGF, TGF-β, lipids, lipid transport proteins, carbohydrates,micronutrients such as minerals, etc.

Chemically Defined Media:  These media contain contamination free  ultra pure inorganic and organic constituents, and may contain pure protein additives, like insulin, epidermal growth factor, etc. that have been produced in bacteria or yeast by genetic engineering with the addition of vitamins, cholesterol, fatty acids and specific amino acids. The CHO cell lines are widely used for being highly stable expression systems for
heterologous genes (those from a different organism), and for its relatively simple adaptation to adherence-independent growth in serum and protein free media.

Protein-Free Media:  In contrast, protein free media do not contain any protein; they only contain non-protein constituents necessary for culture of the cells. The formulations MEM, DME, RPMI-1640,etc. are protein free; where required, protein supplementation is provided.

The animal cell cultures are used for a diverse range of research and development.These areas are:
(a) Production of antiviral vaccines, which requires the standardization  of cell lines for the multiplication and assay of viruses.
(b) Cancer research, which requires the study of uncontrolled cell division in cultures.
(c) Cell fusion techniques.
(d) Genetic manipulation, which is easy to carry out in cells or organ cultures.
(e) Production of monoclonal antibodies requires cell lines in culture.
(f ) Production of pharmaceutical drugs using cell lines.
(g) Chromosome analysis of cells derived from womb.
(h) Study of the effects of toxins and pollutants using cell lines.
(i) Use of artificial skin.
(j) Study the function of the nerve cells.
(k) Many commercial proteins have been produced by animal cell culture and there medical application is being evaluated. Tissue Plasminogen activator (t-PA) was the first drug that was produced by the mammalian cell culture by using rDNA technology. The recombinant t-PA is safe and effective for dissolving blood clots in patients with heart diseases and thrombotic disorders.

Plant tissue culture
Most methods of plant transformation applied to genetically modified crops require that a whole plant is regenerated from isolated plant cells or tissues that have been genetically transformed. This regeneration is conducted in vitro so that the environment and growth medium can be manipulated to ensure a high frequency of regeneration. In addition to this, the regenerable cells must be accessible to gene transfer by whatever technique is chosen. The primary aim is therefore to produce, as easily and as quickly as possible, a large number of regenerable cells that are accessible to gene transfer. The subsequent regeneration
step is often the most difficult step in plant transformation studies

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.
The culture environment When cultured in vitro, all the needs of the plant cells, both chemical 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.

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.
Complete plant cell culture medium is usually made by combining several  different components,
Media components
It is useful to briefly consider some of the individual components of the stock solutions.

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

Plant growth  regulators are the critical media components in determining the developmental pathway of the plant cells. The plant growth regulators used most commonly are plant hormones or their synthetic analogues.
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.
Each class of plant growth regulator will be looked at briefly below.
Auxins promote both cell division and cell growth. The most important naturally occurring auxin is indole-3-acetic acid (IAA), but its use in plant cell culture media is limited because it is unstable to both heat and light. Occasionally, amino acid conjugates of IAA (such as indole-acetyl-l-alanine and indole-acetyl-l-glycine), which are more stable, are used to partially alleviate the problems associated with the use of IAA.
It is more common, though, to use stable chemical analogues of IAA as a source of auxin in plant cell culture media. 2,4-Dichlorophenoxyacetic acid (2,4-D) is the most commonly used auxin and is extremely effective in most circumstances. Other auxins are available and some may be more effective or ‗potent‘ than2,4-D in some instances.
Cytokinins promote  cell division. Naturally occurring cytokinins are a large group of structurally related purine derivatives. Of the naturally occurring cytokinins, two have some use in plant tissue culture media zeatin andN6-(2-isopentyl)adenine (2iP). Their use is not widespread as they are expensive(particularly zeatin) and relatively unstable. The synthetic analogues kinetin and6-benzylaminopurine (BAP) are therefore used more frequently. Non-purine-based chemicals, such as substituted phenylureas, are also used as cytokinins in plant cellculture media. These substituted phenylureas can also substitute for auxin in someculture systems.
There are numerous, naturally occurring, structurally related compounds termed gibberellins. They are involved in regulating  cell elongation, and are agronomically important in determining plant height and fruit-set. Only a few of the gibberellins are used in plant tissue culture media, GA3 being the most common.
Abscisic acid
Abscisic acid (ABA) inhibits cell division. It is most commonly used in plant tissue culture to promote distinct developmental pathways such as somatic embryogenesis
Ethylene is a gaseous, naturally occurring, plant growth regulator most commonly associated with controlling fruit ripening in climacteric fruits, and its use in plant tissue culture is not widespread. It does, though, present a particular problem for plant tissue culture. Some plant cell cultures produce ethylene, which, if it builds up sufficiently, can inhibit the growth and development of the culture. The type of culture vessel used and its means of closure affect the gaseous exchange between the culture vessel and the outside atmosphere and thus the levels of ethylene present in the culture.

Plant growth regulators and tissue culture
Generalizations about plant growth regulators and their use in plant cell culture media have been developed from initial observations made in the 1950s. There is, , some considerable difficulty in predicting the effects of plant growth regulators: this is because of the great differences in culture response among species, cultivars, and even plants of the same cultivar grown under different conditions., some principles do hold true and
have become the paradigm on which most plant tissue culture regimes are based.

Auxins and cytokinins are the most widely used plant growth regulators in plant tissue culture and are usually used together, the ratio of the auxin to the cytokinin determining the type of culture established or regenerated A high auxin to cytokinin ratio generally favours root formation, whereas a high cytokinin to auxin ratio favours shoot formation. An intermediate ratio favours callus production.

Culture types
Cultures are generally initiated from sterile pieces of a whole plant. These pieces are termed  explants, and may consist of pieces of organs, such as leaves or roots, or maybe specific cell types, such as pollen or endosperm. Many features of the explant are known to affect the efficiency of culture initiation. Generally, younger, more rapidly growing tissue (or tissue at an early stage of development) is most effective. Several
different culture types most commonly used in plant transformation studies are as follows.

Explants, when cultured on the appropriate medium, usually with  both an auxin and a cytokinin, can give rise to an unorganized, growing, and dividing mass of cells. It is thought that any plant tissue can be used as an explant, if the correct conditions are found. In culture, this proliferation can be maintained more or less indefinitely, provided that the callus is sub cultured on to fresh medium periodically. During callus formation,
there is some degree of dedifferentiation (i.e. the changes that occur during development and specialization are, to some extent, reversed), both in morphology (a callus is usually composed of unspecialized parenchyma cells) and metabolism. One major consequence of this dedifferentiation is that most plant cultures lose the ability to photosynthesize. This has important consequences for the culture of callus tissue, as the metabolic profile will probably not match that of the donor plant. This necessitates the addition of other components-such as vitamins and, most importantly, a carbon source-to the culture medium, in addition to the usual mineral nutrients. Callus culture is often performed in the dark (the lack of photosynthetic capability being no drawback) as light can encourage differentiation of the callus. During long term culture, the culture may lose the requirement for auxin and/or cytokinin. This process, known as habituation, is common in callus cultures from some plant species(such as sugar beet).
Callus cultures are extremely important in plant biotechnology. Manipulation of the auxin to cytokinin ratio in the medium can lead to the development of shoots, roots, or somatic embryos from which whole plants can subsequently be produced. Callus cultures can also be used to initiate cell suspensions, which are used in a variety of ways in plant transformation studies.

Protoplasts are plant cells with the cell wall removed. Protoplasts are most commonly isolated from either leaf mesophyll cells or cell suspensions, although other sources can be used to advantage. Two general approaches to removing the cell wall (a difficult task without damaging the protoplast) can be taken: mechanical or enzymatic isolation. Mechanical isolation, although possible, often results in low yields, poor quality, and poor performance in culture due to substances released from damaged cells. Enzymatic isolation is usually carried out in a simple salt solution with a high osmoticum, plus the cell-wall-degrading enzymes. It is usual to use a mix of both cellulase and pectinase enzymes, which must be of high quality and purity.
Protoplasts are fragile  and damaged easily, and therefore must be cultured carefully.Liquid medium is not agitated and a high osmotic potential is maintained, at least in the initial stages. The liquid medium must be shallow enough to allow aeration in the absence of agitation.  Protoplasts can be plated out on to solid medium and callus produced. Whole plants can be regenerated by organogenesis or somatic embryogenesis from this callus. Protoplasts are ideal targets for transformation by a variety of means.
Root cultures
Root cultures can be established in vitro from explants of the root tip of either primary or lateral roots and can be cultured on fairly simple media. The growth of roots  in vitro  is potentially unlimited, as roots are indeterminate organs. Although the establishment of root cultures was one of the first achievements of modern plant tissue culture, they are not widely used in plant transformation studies.
Shoot tip and meristem culture The tips of shoots (which contain the shoot apical meristem) can be cultured  in  vitro, producing clumps of shoots from either axillary or adventitious buds. This method can be used for clonal propagation. Shoot meristem cultures are potential alternatives to the more commonly used methods for cereal regeneration as they are less genotype dependent and more efficient (seedlings can be used as donor material).
Embryo culture
Embryos can be used as explants to generate callus cultures or somatic embryos. Both immature and mature embryos can be used as explants. Immature, embryo-derived embryogenic callus is the most popular method of monocotyledon plant regeneration.

Food Biotechnology  by K.V.Anand Raj & M. Raveendra Reddy