PLANT ORGAN, CALUS CULTURE AND CELL SUSPENSION CULTURE

SBT 2131LECTURE 3

ASSOC. PROF. DR. AROKIARAJ

ORGAN CULTURES

In vitro aseptic culture of isolated organs of plants such as anthers, ovaries, roots, shoots or other organs on suitable nutrient media for induction of callus for somatic embryogenesis or alternatively for direct regeneration.

CALLUS CULTURES

Callus cultures are usually initiated from tissues or organ explants after varying periods of incubation on agar culture media.

Most frequently, the vacuolated parenchymatous cells of the pith, cortex or mesophyll are stimulated to divide.

Highly differentiated cells at the periphery of the explant divide in response to wounding or from the influence of natural or synthetic growth hormones supplied exogenously in the culture medium.

The callus cells show an increase in cytoplasmic activity, followed by a rise in protein synthesis and respiration rate.

Successive division result in the cells becoming small and cytoplasmically dense.

CALLUS CULTURES

At the meristematic state, a phenomenon known as de-differentiation occurs where the explant increases in size as new cells are formed by mitosis and cytokinesis.

Cell division and cell expansion eventually result in the formation of callus tissue over the surface and the cut ends of the explant.

When large enough to handle, this tissue is excised from the explant or transferred to fresh medium.

The callus is then maintained indefinitely by sub-cultures at regular intervals.

CALLUS CULTURES

Growth rate of cells from different species:

Generally, three types of growth rates are experienced by plant cells in tissue culture.

Slow growing cells (it takes about 1 month for the cells to divide)

Medium growing cells (this takes about 2 weeks for the cells to divide) and

Fast growing cells (this takes about 10 days for the cells to divide)

MORPHOLOGY AND STRUCTURE OF CALLUS TISSUES

Calli proliferate as irregular masses of tissue (in a disorganized fashion), and vary considerably in growth rates, appearance and texture.

Some are soft (minimal cellular contact),Some are hard (closely packed cells) and Some are friable, and grow as a single mass of cells.

Others are compact and may have a nodular appearance.

CALLUS CULTURE

Callus developing on a stem segment explant

Callus developing from anthers

Callus developing from cotyledon explants

Callus developing from seed integuments

Callus developing from roots

CALLUS CULTURE

For example, callus of onion (Allium cepa) producesconsiderable amounts of surface mucilage, giving a glisteningappearance.

This indicates that the morphology and growth characteristicsof a tissue are related to the composition of the culture medium,particularly the levels of growth hormones and the explant usedto initiate the culture and the particular cultivar.

Even tissues initiated from different explants from the samecultivar may show considerable variation in morphology.

Most callus are creamy-yellow in colour, whether induced indarkness or under diffuse light.

Others develop chlorophyll and carotenoids in plastids.

HABITUATED CALLUS CULTURES

Most callus tissues require growth regulators in the culture medium to induce cell proliferation.

However, cells of well established callus tissues sometimes undergo spontaneous change after prolonged sub-culture (reflected by alteration in their requirements for exogenous hormone). These are classed as habituated callus and they continue to grow after transfer to medium without auxins or cytokinins.

Fully habituated callus are those which grow in the absence of both growth regulators.

HABITUATED CALLUS CULTURES

Such callus tissues can be easily distinguished from hormone-requiring cells by a change in their morphology and texture.

Causes of habituation:

-may be related to enzymatic alterations that cause changes in metabolic and nutritional requirements,

-genetic changes such as somatic mutation and increased ploidy, that affect auxin-regulating systems.

CELLULAR TOTIPOTENCY

The cloning of isolated single cells in vitro has demonstrated the fact that somatic cells, under appropriate conditions has the ability to regenerate to complete plantlets.

Thus the potential of a single cell to grow and develop a multi-cellular or multi-organised higher organism is termed totipotency.

The potential therefore lies mainly in cellular differentiation. This indicates that all the genes responsible for differentiation are present within the individual cells and many of them remain inactive in differentiated tissues or organs are able to express only under adequate culture conditions.

Therefore, to express totipotency, the differentiated cell first undergoes dedifferentiation and then redifferentiation.

CELLULAR TOTIPOTENCY

This phenomenon of a mature cell reverting to a meristematic state and forming undifferentiated callus tissue is termed dedifferentiation.

Whereas the ability of a dedifferentiated cell to form a whole plant or plant organs is termed redifferentiation.

The basic development is cell differentiation and this is referred to as cyto-differentiation.

SCHEME SHOWING CYTO-DIFFERENTIATION IN PLANT CELLS

Isolated cells from explants undergoes dedifferentiation to form callus. Callus cells then redifferentiate to form embryoids (somatic embryogenesis) which then develops to form shoots and roots (plantlet regeneration).

TYPES OF CULTURES FAVOURABLE FOR DIFFERENTIATION

Callus cultures: Stable characteristic (fairly homogeneous mass of cells, can be proliferated in large amounts under known culture conditions) after subculture. However, many cultures lose their potential for differentiation during continual subculture due to epigenetic changes.

Suspension cultures: Cells receive more hormogenous stimuli; Synchrony of differentiation is higher in such cells; Enzymes associated with differentiation enhanced in such cells

Single cells : Ideal for investigating cellular differentiation; Mesophyll cells of certain plants are able to differentiate into tracheary elements within 3 days in a culture medium with 1 ppm 2,4-D and 1ppm Kinetin

TYPES OF CULTURES FAVOURABLE FOR DIFFERENTIATION

Protoplast culture: Plant cells without cell wall able to form micro-callus with cell wall capable of differentiation in the presence of auxin and cytokinin.

CELL SUSPENSION CULTURES

Suspension Cultures: Initiated by transferring established callus tissues to a liquid medium which is then agitated by shaking. After 2 or 3 weeks a suspension of actively growing cells is produced.

A friable callus is normally used to generate suspension cultures

A less friable callus can also be used by modifying the culture medium.

No suspension culture has yet been shown to be composed entirely of free floating cells (they consist of aggregate and single cells). This contributes to non-uniformity of cell size, shape and metabolism (a characteristic of cell suspension)

GROWTH CURVE OF BATCH-PROPAGATED SUSPENSION CULTURE

The plot of cell number against time shows a distinct pattern of growth curve characteristic by five phases.

Inoculation is followed by a lag phase (when cells in the fresh medium prepare to divide).

Cells then undergo a short exponential growth phase (where the rate of division is minimal).

Followed by a linear growth phase.

Cell division then slows down (during progressive deceleration phase)

During stationary phase (cell numbers in a culture medium remains more or less constant)

MORPHOLOGY OF CELLS IN SUSPENSION

Generally, suspension cultures consist of varying proportions of cell aggregates and free (single) cells.

There is often considerable variation in the morphological appearance of the free cells in a single culture. Abnormal cell-shapes are often correlated with changes in the chromosome complement of the cells such as increase in ploidy.

Again, as in callus cultures, the size and nature of the aggregates depends to some extent on the composition of the culture medium, particularly the type and concentration of growth hormones present.

Cells therefore undergo characteristic patterns of morphological and cytological change during batch propagation, reflecting the change in the nutrient environment.

MORPHOLOGY OF CELLS IN SUSPENSION

Upon sub-culture of the suspension to fresh medium, the resumption of cytoplasmic activity and the appearance of prominent cytoplasmic strands are observed.

Organelles such as mitochondria and plastids can be observed in the cytoplasm.

Cell division then results in the formation of chains of light aggregates of cells. Cytoplasmic activity declines as division slows down and the cells expand and separate as they again enter stationary phase.

PHYSIOLOGICAL ASPECTS OF CELLULAR TOTIPOTENCY

Hormonal Control

Phytohormones particularly auxins are reported to affect vascular differentiation quantitatively and qualitatively. Some evidence also points towards the involvement of cytokinins and gibberellins in the process of xylogenesis.