Tissue culture

WHAT IS TISSUE CULTURE?

It is a technique of growing cells, tissue, organs or whole organism in vitro on an artificial culture media in an aseptic and controlled condition.

Tissue culture involves the use of small pieces of plant tissue (explants) which are cultured in a nutrient medium under sterile conditions. Non-dividing parenchyma, cells of meristem, vascular cambial tissues and embryonic tissues are at early stage of development and exist in an "undetermined" state which rapidly proliferate and produce callus.

CULTURE MEDIA:

Components of a typical culture media includes:

• Organic nitrogen sources (e.g., glycine, inositol)
• Carbon source (e.g., glucose , sucrose)
• Inorganic micronutrients (e.g., Mn, Cu, Zn, B, Na, Cl, I, S, Mo, Co, Al, Ni)
• Inorganic macronutrients (e.g., Fe, Mg, Ca, K, P, N)
• Vitamins (e.g., Nicotinic acid, pyridoxine, thiamine)
• Plant growth regulators (e.g., Auxin, cytokinin)
• Antibiotics (e.g., Kanamycin)
• If media is semi-solid then a good quality gelling agent (Agar)
• Example - MS media, B5 media 

TYPES OF CULTURE MEDIA:

On the basis of consistency of the media, they are of two types:

1. Solid Media/ Semi-solid media: The usual gelling agent for solid or semisolid medium is agar, a hydro colloid derived from red algae.
2. Liquid media: It is also called as broth and contains only dissolved nutrients in water. Liquid media are used for growth of pure batch cultures for fermentation studies and various other purposes while solidified media can be used widely for isolation of pure cultures for estimating viable microbial population and a variety of other purposes.

PREPARATION OF EXPLANTS:

• Meristems are actively mitotically dividing usually free of viruses infection because they lack vascular tissue which transports viruses.
• The smallest possible meristem explants should be taken in order to avoid infection.
• Heat treatment (34-35C) to meristem explants is commonly used to inactivate the virus to reduce the possibility of infection.
• The explants are first sterilized. Common sterilizing agents are 1-2% sodium hypochlorite, 9-10% calcium hypochlorite (10-40 min., young stems, petioles, roots, fruits) and 70% ethanol (2-5 min.. leaves, seeds)

SEED 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:

Embryo culture is the type of tissue culture that involves the isolation of an embryo from  a given organism for in vitro growth.

Embryo culture may involve the use of a mature or 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.

CALLUS CULTURE:

This 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.

ORGAN CULTURE:

Organ culture is the 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 in 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.

MERISTEM CULTURE:

Cultivation of axillary or apical shoot meristem, particularly of shoot apical meristem, is known as meristem culture.

PROTOPLAST CULTURE:

Protoplasts are naked plant cells without the cell wall, but they possess plasma membrane and all other cellular components. They represent the functional plant cells but for the lack of the barrier, cell wall. Protoplasts of different species can be fused to generate a hybrid and this process is referred to as somatic hybridization (or protoplast fusion).

PLANT REGENERATION FROM CALLUS:

• The relative level of auxin and cytokinin controls the shoot and root formation.
• A callus can be regenerated into a whole plant by simply altering the concentration of growth regulators.
• When auxin/cytokinin ratio is high, only roots are formed.
• When cytokinin/auxin ratio is high, shoots are formed.
• When levels of both hormones are intermediate, it results in completely disorganized callus growth.

SOMATIC HYBRIDIZATION:

Somatic fusion, also called protoplast fusion, is a type of genetic modification in plants by which two distinct species of plants are fused together to form a new hybrid plant with the characteristic of both, a somatic hybrid.

MICROPROPAGATION:

Micropropagation involves in vitro propagation of the selected genotype and ultimate establishment of the plant in the field or a glasshouse.

There are five stages in micropropagation:

1. Preparation of explant : the plant material for in vitro culture is prepared and explants are obtained which requires less aggressive sterilization.
2. Formation of callus
3. Shoot proliferation: a high cytokinin/auxin ratio induces shoot formation and high auxin/cytokinin ratio induces root formation. After 4-8 weeks, the original explant is transformed into a mass of branched shoots or a cluster of basal shoots. The small shoots or clusters are then re-planted on to a fresh medium (in which the cytokinin level can be increased) to increase shoot multiplication.
4. Shoot elongation and root formation: Adventitious and axillary shoots lack roots in the presence of cytokinin, fresh liquid media added to established cultures (double layer technique). Auxins such as NAA are usually required to induce rooting and activated charcoal can be added to the liquid medium in order to adsorb any residual cytokinin.
5. Transfer to a glasshouse: The micro propagated plantlet must acclimatize to the environment of the glasshouse with appropriate humidity and temperature control. Acclimatization can proceed in vitro with bottom cooling, reducing the relative humidity in the head space of the container. The culture vessels are uncapped and placed in the glasshouse several days prior to the removal of the plants from the culture medium.

PLANT REGENERATION PATHWAYS:

• Organogenesis: It is the process of morphogenesis involving the formation of plant organs i.e., shoots, roots, flowers, buds from explant or cultured plant tissues.
• Somatic embryogenesis: The process of regeneration of embryos from somatic cells, tissues or organs is regarded as somatic (or asexual) embryogenesis. Somatic embryogenesis may result in non-zygotic embryos or somatic embryos (directly formed from somatic organs), pathogenetic embryos (formed from unfertilized egg) and androgenic embryos (formed from male gametophyte).

SOMACLONAL VARIATION ('SPORTS')

It refers to genetic variations in plants that have been produced by plant tissue culture and can be detected as genetic or phenotypic traits.

Progeny that differ significantly from the parent are called 'somatic variants'. These somatic variants have established new and important varieties such as the naval orange or nectarines. Potato and sugarcane 'sports' were found to be resistant to disease in contrast to the susceptible parent plants. Herbicide resistance, cold- and salt- tolerance are additional favorable traits that can be found in 'sports'.