ESTABLISHMENT AND MAINTENANCE OF CALLUSES

ESTABLISHMENT AND MAINTENANCE OF CALLUSES

For rising the callus tissues, a tissue culturist must have clear understanding of some basic principles. A cell from any part of the part of the plant like shoot apex, bud, leaf, mesophyll cells, epidermis, cambium, anthers, pollen, fruit etc., when inoculated in a suitable medium under aseptic laboratory conditions can able to differentiate and multiply. This results into the formation of an amorphous mass of cells known as callus, which can be induced to re-dfferentiate on appropriate medium to develop embryoids which directly develop into the plantlets, eventually giving rise to a whole viable plant. Isolated cells from differentiated tissues are generally non dividing and quiescent but to express totipotency, the differentiated cells first undergoes de-differentiation and then    re-differentiation.

Dedifferentiation: The phenomenon of a mature cell reverting to a meristematic state and forming a dedifferentiated callus tissue

Redifferentiation is the ability of dedifferentiated cell to form whole plant/plant organs is termed as re-differentiation.

Callus induction, subculture and maintenance

Callus

Callus (pl. Calli) on a wounded plant parts or on a culture medium is made of an amorphous aggregate of loose parenchyma cells which proliferate from the mother cells. Callus is either homogenous parenchymatous mass or treachery elements or sieve elements or submerized cells or secretory cells or the trichomes. Calus is somewhat an abnormal tissue which has the potentiality to produce normal roots and embryoids and in turn it develops into plantlets. Callus may be hard (due to lignification of cell walls) or brittle and sometimes soft.

For callus initiation, transfer explants to the required semi solid medium aseptically and gently press them into the agar so that good contact is made. Radical tips will callus well if laid horizontally on the agar whereas stem section may produce more callus if placed vertically with one cut end in the agar.

Within 2-3 weeks, explants should show new growth as pustules or protuberances or as a fine mat across the surface depending on the distribution and mitotic activity of the parenchyma residing in the excised tissues. When cultured for several weeks, any callus will show signs of ageing, noted as deceleration of growth, necrotic or browning and finally desiccation. The newly formed callus will be removed from the initial explant at this stage by cutting with the sterile scalpel. Once well established, most callus cultures will require regular subculture at approximately 4 weekly intervals.

Cell suspension culture

The term "suspension culture" has simply aggregates of cells suspended in liquid medium. After few subcultures, samll bits from a soft callus can be cut and inoculated into liquid medium where they give rise to a suspension culture. Agitation rates on orbital shakers should be in the range of 30-150 rpm. The degree of cell separation of established cultures of high friability can be modified by changing the composition of the nutrient medium particularly the concentration of growth regulators.

Subculture and Measurement of Growth

Cell suspension usually requires regular subculture at more frequent intervals than callus growth from which they are derived. Subculture involves the aseptic transfer of suitable size inoculums to fresh medium using either pipettes (or) autoclavable metal syringes, and transfer by simply tipping culture into the new vessel up to a graduation mark to ensure approximately constant inoculum’s size. The latter method is only one practicable with cultures containing large cell aggregates.

Secondary metabolites production in cell lines

Higher plants are valuable sources of industrially important natural products which include flavors, fragrances, essential oils, pigments, sweeteners, feed stocks, anti microbial and pharmaceuticals which belong to metabolic groups collectively referred to as secondary metabolites. These substances do not participate in vital metabolic functions of the host plant tissues in the same manner as amino acids, nucleic acids or other primary metabolites but appear to serve as a chemical interface between the producing plant and its surrounding environment. These are used to ward off predators and to attract pollinators. They may also help in combating infectious diseases.

Advantages of secondary metabolites

Ø  Plant cells are relatively easy to grow.

Ø  The rate of cell growth and biosynthesis in cultures initiated from a very small amount of plant material is quite high and the final product may be produced in a considerably short period of time.

Ø  Plant cell cultures are maintained under controlled environmental and nutritional conditions which ensure continuous yields of metabolites.

Ø  Suspension cultures offer a very effective way of incorporating precursors into cells

Ø  New routes of synthesis can be recovered from deviant and mutant cell lines which can lead to production of novel compounds not previously found in whole plants.

Ø  Some cell cultures have the capacity for bio transformation of specific substrates to more valuable products by means of single or multiple step enzyme activity.

Ø  Culture of cells may be more economical for those plants which take long period to achieve maturity.