Tissue culture methods and applications
Tissue culture refers to a method in which fragments of a tissue (plant or animal tissue) are introduced into a new, artificial environment, where they continue to function or grow. While fragments of a tissue are often used, it is important to note that entire organs are also used for tissue culture purposes. Here, such growth media as broth and agar are used to facilitate the process.
Methods of Tissue Culture
Seed culture is the type of tissue culture that is primarily used for plants such as orchids. For this method, explants (tissue from the plant) are obtained from an in-vitro derived plant and introduced in to an artificial environment, where they get to proliferate. In the event that a plant material is used directly for this process, then it has to be sterilized to prevent tissue damage and ensure optimum regeneration.
Embryo culture is the type of tissue culture that involves the isolation of an embryo from a given organism for in vitro growth. *Note, the term embryo culture is used to refer to sexually produced zygotic embryo culture. Embryo culture may involve the use of a mature of immature embryo. Whereas mature embryos for culture are essentially obtained from ripe seeds, immature embryo (embryo rescue) involves the use of immature embryos from unripe/hybrid seeds that failed to germinate. In doing so, the embryo is ultimately able to produce a viable plant.
For embryo culture, the ovule, seed or fruit from which the embryo is to be obtained is sterilized, and therefore the embryo does not have to be sterilized again. Salt sucrose may be used to provide the embryo with nutrients. The culture is enriched with organic or inorganic compounds, inorganic salts as well as growth regulators.
Callus is the term used to refer to unspecialized, unorganized and a dividing mass of cells. A callus is produced when explants (cells) are cultured in an appropriate medium – A good example of this is the tumor tissue that grows out of the wounds of differentiated tissues/organs.
In practice, callus culture involves the growth of a callus (composed of differentiated and non- differentiated cells), which is the followed by a procedure that induces organ differentiation.
For this type of tissue culture, the culture is often sustained on a gel medium, which is composed of agar and a mixture of given macro and micronutrients depending on the type of cells. Different types of basal salt mixtures such as murashige and skoog medium are also used in addition to vitamins to enhance growth.
Organ culture is a type of tissue culture that involves isolating an organ for in vitro growth. Here, any organ plant can be used as an explant for the culture process (Shoot, root, leaf, and flower).
With organ culture, or as is with their various tissue components, the method is used for preserve their structure or functions, which allows the organ to still resemble and retain the characteristics they would have in vivo. Here, new growth (differentiated structures) continues given that the organ retains its physiological features. As such, an organ helps provide information on patterns of growth, differentiation as well as development.
Major Steps of Tissue Culture method (Plants)
Initiation Phase (Stage 1)
The initiation phase is the first phase of tissue culture. Here, the tissue of interest is obtained and introduced and sterilized in order to prevent any microorganism from negatively affecting the process. It is during this stage that the tissue is initiated in to culture.
Multiplication Phase (Stage 2)
The multiplication phase is the second step of tissue culture where the in vitro plant material is re- divided and then introduced in to the medium. Here, the medium is composed of appropriate components for growth including regulators and nutrients. These are responsible for the proliferation of the tissue and the production of multiple shoots.
*This step is often repeated several times in order to obtain the desired number of plants
Root formation (Stage 3)
It is at this phase that roots are formed. Here, hormones are required in order to induce rooting, and consequently complete plantlets.
Applications of tissue culture
Rapid Clonal Propagation
A clone is a group of individuals or cells derived from a single parent individual or cell through asexual reproduction. All the cells in callus or suspension culture are derived from a single explants by mitotic division. Therefore, all plantlets regenerated from a callus/suspension culture generally have the same genotype and constitute a clone. These plantlets are used for rapid clonal propagation.
Genetic variation present among plant cells of a culture is called soma-clonal variation. The term soma-clonal variation is also used for the genetic variation present in plants regenerated from a single culture. This variation has been used to develop several useful varieties.
A gene that is transferred into an organism by genetic engineering is known as transgene. An organism that contains and expresses a transgene is called transgenic organism. The transgenes can be introduced into individual plant cells. The plantlets can be regenerated from these cells. These plantlets give rise to the highly valuable transgenic plants.
Induction and Selection of Mutations
Mutagens are added to single cell liquid cultures for induction of mutations. The cells are washed and transferred to solid culture for raising mu ant plants. Useful mutants are selected for further breeding. Tolerance to stress like pollutants, toxins, salts, drought, flooding etc. can also be obtained by providing them in culture medium in increasing dosage. The surviving healthy cells are taken to solid medium for raising resistant plants.
Resistance to Weedicides
It is similar to induction of mutations. Weedicides are added to culture initially in very small concentrations. Dosage is increased in subsequent cultures till die desired level of resistance is obtained. The resistant cells are then regenerated to form plantlets and plants.