TISSUE CULTURE:Techniques & Uses

What is Tissue Culture?

Tissue culture is a process that involves exposing plant tissue to a specific regimen of nutrients, hormones, and light under sterile, in vitro conditions to produce many new plants, each a clone of the original mother plant, over a very short period of time. AgriForest's tissue culture plants are characterised by disease free growth, a more fibrous, healthier root system, a bushier branching habit, and a higher survival rate.

There are three main steps to the tissue culture process:

STAGE I is the initiation phase. It concerns the establishment of plant tissue in vitro by sterilising the material and initiating it into culture.

STAGE II is the multiplication phase. At this stage, the in vitro plant material is re-divided and placed in a medium with plant growth regulators that induce the proliferation of multiple shoots. This process is repeated many times until the number of plants desired is reached.

STAGE III is the root formation phase. It involves the introduction of hormones to induce rooting and the formation of complete plantlets.

Carrel (1912) first succeeded in growing tissue explants for many cell generations. Since then considerable progress has been made in the techniques of tissue culture. In early days of tissue culturing the technique consisted of explanting small portions of different tissues (preferably embryonic) in a medium consisting of blood serum and chick embryo extract, plus saline solution.

After 1955 first chemically defined culture media became available. For example, Parker’s 199 synthetic medium contains many different metabolites. Some foetal calf surum or horse serum is also added to produce cell growth. Most cells of eukaryotes can be grown with the addition of a small percentage of serum in synthetic media.

Cell cultures are required for the modern work in biochemistry, molecular biology and cell physiology. The most commonly grown cells are bacteria or mammalian tissue cells, although amphibian cells and plant cells are now being used more extensively.

Thus it is now possible to grow cells and tissues outside of the body of organisms and under laboratory conditions with ease. Cell culture benefits the cytologist and pathologist. Variety of tissues from primate group such as bone marrow, skin, spleen, kidney and testis has been studied by tissue culture. A general scheme of tissue culture for cytological study is given here.

Method of tissue culture:

A few tissue pieces are collected from a living subject and placed in a sterile chamber with a physiological saline solution for washing and brief storage. The cells are dissociated from each other by treatment with an enzyme (Trypsin) in a low concentration (about 0.2%) in a buffered solution.

After dissociation of the cells by trypsinization, the cell suspension is centrifuged and suspended in a special medium containing all the nutritional requirements for cell division and maintenance.

The cells suspended in the growth medium are placed in Leighton tubes and incubated at 37°C. A depression in the Leighton tube allows for the introduction of a cover slip, to which cells may adhere. Subcultures are made by taking small samples from the tubes and placing them in other tubes with appropriate medium.

Accumulation of larger number of dividing cells can be increased by application of 5-amino uracil or excess thymidine before colchicine treatment. Staining is by Feulgen reaction, acetocarmine or acetoorcein.

1. Explant method:

For growing avian or mammalian cells. A small fragment of tissue is placed on a coverslip and covered with a drop of chick plasma and growth medium. The plasma sets to form a clot. The cover ship is mounted in a cavity slide with the clot attached to the under surface. The space in the cavity provides a source of oxygen and allows for exchange of CO₂. The cells grow out from the tissue fragment as a sheet within the plasma clot.

2. Monolayer method:

It is used for preparing large numbers of cells in culture. The chopped tissue is first treated with dilute trypsin for breaking the intercellular cement and thus a suspension of separated cells is produced. Trypsin is then removed by washing in a centrifuge and cell suspension is mixed with culture medium.

The cells settle on the glass, attach to the surface of the vessel and grow to form a monolayer. When culture has covered the glass surface, cells are again treated with trypsin for separation. They can then be used to prepare fresh monolayers.

3. Organ culture method:

It is used to preserve in vivo characteristics of cells and tissues. It is used for fragments of tissue about 1.5 mm size. These fragments are placed on a raft or permeable sheet such as Millipore material as in Trowell method.

Uses of Plant tissue Culture-

Plant tissue culture now has direct commercial applications as well as value in basic research into cell biology, genetics and biochemistry. The techniques include culture of cells, anthers, ovules and embryos on experimental to industrial scales, protoplast isolation and fusion, cell selection and meristem and bud culture. Applications include:

• micropropagation using meristem and shoot culture to produce large numbers of identical individuals
• screening programmes of cells, rather than plants for advantageous characters
• large-scale growth of plant cells in liquid culture as a source of secondary products
• crossing distantly related species by protoplast fusion and regeneration of the novel hybrid
• production of dihaploid plants from haploid cultures to achieve homozygous lines more rapidly in breeding programmes
• as a tissue for transformation, followed by either short-term testing of genetic constructs or regeneration of transgenic plants
• removal of viruses by propagation from meristematic tissues

shailesh kr shukla

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