PLANT ORGAN, CALUS CULTURE AND

CELL SUSPENSION CULTURE

SBT 2131
LECTURE 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) produces
considerable amounts of surface mucilage, giving a glistening
appearance.

This indicates that the morphology and growth characteristics

of a tissue are related to the composition of the culture medium,
particularly the levels of growth hormones and the explant used
to initiate the culture and the particular cultivar.

Even tissues initiated from different explants from the same

cultivar may show considerable variation in morphology.

Most callus are creamy-yellow in colour, whether induced in

darkness 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.