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Tissue Culture

Tissue culture is the growth of tissues and/or cells separate from the organism. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar. Tissue culture commonly refers to the culture of animal cells and tissues, while the more specific term plant tissue culture is used for plants. Tissue culture produces clones, in which all product cells have the same genotype (unless affected by mutation during culture)

Historical usage
In 1885 Wilhelm Roux removed a portion of the medullary plate of an embryonic chicken and maintained it in a warm saline solution for several days, establishing the basic principle of tissue culture. In 1907 the zoologist Ross Granville Harrison demonstrated the growth of frog nerve cell processes in a medium of clotted lymph.

Modern usage
In modern usage, "tissue culture" generally refers to the growth of eukaryotic cells in vitro. It is often used interchangeably with cell culture to specifically describe the in vitro culturing of sperm donor cells. However, "tissue culture" can also be used to refer to the culturing of tissue pieces, i.e. explant culture or whole organs, i.e. organ culture. It is a tool for the study of animal cell biology in vitro model of cell growth to allow a highly selective environment which is easily manipulated (used to optimize cell signaling pathways). In 1920 germen scientist Harbland is considered as the father of tissue culture.

Tissue culture, both plant and animal has several critical requirements
1. Appropriate tissue (some tissues culture better than others) 2. A suitable growth medium containing energy sources and inorganic salts to supply cell growth needs. This can be liquid or semisolid 3. Aseptic (sterile) conditions, as microorganisms grow much more quickly than plant and animal tissue and can over run a culture 4. Growth regulators - in plants, both auxins & cytokinins. In animals, this is not as well defined and the growth substances are provided in serum from the cell types of interest 5. Frequent sub culturing to ensure adequate nutrition and to avoid the buildup of waste metabolites

Classification
Tissue culture may be of two types based on medium. 1. Plant tissue culture 2. Animal tissue culture

Plant Tissue Culture


Plant tissue culture is a practice used to propagate plants under sterile conditions, often to produce clones of a plant. Different techniques in plant tissue culture may offer certain advantages over traditional methods of propagation, including: a) The production of exact copies of plants that produce particularly good flowers, fruits, or have other desirable traits. b) To quickly produce mature plants. c) The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds. d) The regeneration of whole plants from plant cells that have been genetically modified. e) The production of plants in sterile containers that allows them to be moved with greatly reduced chances of transmitting diseases, pests, and pathogens. f) The production of plants from seeds that otherwise have very low chances of germinating and growing, i.e.: orchids and nepenthes. g) To clean particular plant of viral and other infections and to quickly multiply these plants as 'cleaned stock' for horticulture and agriculture. Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant (totipotency). Single cells, plant cells without cell walls (protoplasts), pieces of leaves, or (less commonly) roots can often be used to generate a new plant on culture media given the required nutrients and plant hormones.

Culturing (micro propagating) Plant Tissue - the steps


1. Selection of the plant tissue (explant) from a healthy vigorous mother plant - this is often the apical bud, but can be other tissue 2. This tissue must be sterilized to remove microbial contaminants

3. Establishment of the explant in a culture medium. medium sustains the plant cells and encourages division. It can be solid or liquid 4. Each plant species (and sometimes the variety within a species) has particular medium requirements that must be established by trial and error 5. Multiplication- The explant gives rise to a callus (a mass of loosely arranged cells) which is

The cell

manipulated by varying sugar auxin (low): cytokinin (high) ratios to

concentrations and form multiple shoots

the

6. The callus may be subdivided a number of

times

7. Root formation - The shoots are transferred to a growth medium with relatively higher auxin: cytokinin ratios 8. The rooted shoots are potted up (deflasked) and hardened off by gradually decreasing the humidity 9. This is necessary as many young tissue culture plants have no waxy cuticle to prevent water loss

Why plant tissue culture is needed


1. A single explant can be multiplied into several thousand plants in less than a year - this allows fast commercial propagation of new cultivars 2. Taking an explant does not usually destroy the mother plant, so rare and endangered plants can be cloned safely 3. Once established, a plant tissue culture line can give a continuous supply of young plants throughout the year 4. In plants prone to virus diseases, virus free explants (new meristem tissue is usually virus free) can be cultivated to provide virus free plants 5. Plant tissue banks can be frozen, then regenerated through tissue culture 6. Plant cultures in approved media are easier to export than are soil-grown plants, as they are pathogen free and take up little space (most current plant export is now done in this manner) 7. Tissue culture allows fast selection for crop improvement - explants are chosen from superior plants, then cloned 8. Tissue culture clones are true to type as compared with seedlings, which show greater variability

Applications of Plant Tissue Culture


Plant tissue culture is used widely in plant science; it also has a number of commercial applications. Applications include: a) Micropropagation is widely used in forestry and in floriculture. Micropropagation can also be used to conserve rare or endangered plant species. b) A plant breeder may use tissue culture to screen cells rather than plants for advantageous characters, e.g. herbicide resistance/tolerance. c) Large-scale growth of plant cells in liquid culture inside bioreactors as a source of secondary products, like recombinant proteins used as biopharmaceuticals. d) To cross distantly related species by protoplast fusion and regeneration of the novel hybrid. e) To cross-pollinate distantly related species and then tissue culture the resulting embryo which would otherwise normally die (Embryo Rescue). f) For production of doubled monoploid (dihaploid) plants from haploid cultures to achieve homozygous lines more rapidly in breeding programmes, usually by treatment with colchicine which causes doubling of the chromosome number.

g) As a tissue for transformation, followed by either short-term testing of genetic constructs or regeneration of transgenic plants. h) Certain techniques such as meristem tip culture can be used to produce clean plant material from virused stock, such as potatoes and many species of soft fruit. i) micropropagation using meristem and shoot culture to produce large numbers of identical individuals

Animal Tissue Culture


The term tissue culture refers to the culture of whole organs, tissue fragments as well as dispersed cells on a suitable nutrient medium. It can be divided into (1) organ culture and (2) cell culture mainly on the basis of whether the tissue organisation is retained or not. The beginning of animal tissue culture can be traced to 1880 when Arnold showed that leucocytes can divide outside the body. Later, in 1903, Jolly studied the behaviour of animal tissue explants immersed in serum, lymph or ascites fluid. In 1907, Harrison cultured frog tadpole spinal chord in a lymph drop hanging from a cover slip into the cavity on a microscopic slide; this is regarded as the turning point. A few years later, in 1913, Carrel developed a complicated methodology for maintaining cultures free from contamination, especially by bacteria. Subsequently, suitable culture media were developed and the techniques of cell culture were refined.

Culturing Animal Tissues- The steps


1. Animal tissue is obtained either from a particular specimen, or from a tissue bank of cryo-preserved (cryo = frozen at very low temperatures in a special medium) 2. Establishment of the tissue is accomplished in the required medium under aseptic conditions 3. Growing the cells / tissue requires an optimum temperature, and subculturing when required 4. Human cells, for example are grown at 37degrees and 5% CO2

Animal tissue/cell culture - differences from plant tissue culture


1. Animal cell lines have limited numbers of cell cycles before they begin to degrade 2. Animal cells need frequent subculturing to remain viable 3. Tissue culture media is not as fully defined as that of plants - in addition to inorganic salts, energy sources, amino acids, vitamins, etc., they require the addition of serum (bovine serum is very common, but others are used) 4. Animal tissue cultures can pose biohazard concerns, and cultures require special inactivation with hypochlorite (e.g. Janola,Chlorox, etc.) and then incineration

Uses of Animal Tissue Culture


1. Growing viruses - these require living host cells 2. Making monoclonal antibodies, used for diagnosis and research 3. Studying basic cell processes 4. Genetic modification & analysis 5. Knockout technology - inactivating certain genes and tracing their effects 6. Providing DNA for the Human Genome Project (and other species genome projects)

Bibliography
1. Dodds, J.H., Roberts, L.W., 1995, Experiments in Plant Tissue Culture, 3rd ed., Cambridge University Press 2. Hartmann, H., Kester, D., et.al., 1997, Plant Propagation, 6th ed., Prentice Hall International 3. http://www.une.edu.au/agronomy/AgSrHortTCinfo.html 4. http://aggie-horticulture.tamu.edu /tisscult/pltissue/pltissue.html 5. http://www.liv.ac.uk/~sd21/tisscult/what.htm 6. http://user.school.net.th/~anoparp/bptc1.htm