The starting point for all tissue cultures is plant tissue, called an
explant. It can
be initiated from any part of a plant—root, stem, petiole, leaf, or
although the success of any one of these varies between species. It is
that the surface of the explant is sterilized to remove all microbial
Plant cell division is slow compared to the growth of bacteria and
even minor contaminants will easily overgrow the plant tissue culture.
explant is then incubated on a sterile nutrient medium to initiate the
culture. The composition of the growth medium is designed to both
plant cells, encourage cell division, and control development of either
undifferentiated cell mass, or particular plant organs.
The concentration of the growth regulators in the medium, namely auxin
and cytokinin, seems to be the critical factor for determining whether a
culture is initiated, and how it subsequently develops. The explant
initially form a callus, from which it is possible to generate multiple
and then shoots, forming the basis for plant regeneration, and thus the
of micropropagation. The first stage of tissue culture initiation is
information on what combination of media components will produce a
fast-growing callus, or a green chlorophyllous callus, or embryo, root,
There is at present no way to predict the exact growth medium, and
protocol, to generate a particular type of callus. These
characteristics have to be
determined through a carefully designed and observed experiment for
plant species, and frequently also for each new variety of the species
taken into tissue culture. The basis of the experiment will be media
that give the desired effect in other plant species, and experience.
The strategy for designing a medium to initiate tissue culture, showing
growth regulators and other factors modulate development, can be
using the African violet, a popular house plant. Leaf sections are the
explants. This demonstration is regularly carried out by a student
produces reliable results. Sterile supplies are provided from central
and provision of sterile working areas (for example, in laminar flow
an advantage, although cultures can be initiated in an open laboratory
careful aseptic technique. The standard precautions used during any
work involving chemicals or microbes should be adopted. If you are in
doubt about safety hazards associated with this demonstration, you
consult your local safety advisor.
Step 1: Selection of the Leaves
Leaves are cut from healthy plants, leaving a short length of petiole
They should be selected to each yield several explants of leaf squares,
approximately 1-cm sides. The youngest and oldest leaves should be
Wash the dust off the leaves in a beaker of distilled water, holding the leaf
stalk with forceps.
Step 2: Surface Sterilization and Preparation of the Explants
This part of the procedure should be carried out in a sterile working area, or
with meticulous aseptic technique.
The leaf, with the petiole still attached, should be immersed in 70% ethanol
for 30 seconds, then transferred to a sterile petri dish. Sterile scissors and
forceps are then used to cut squares from the leaf as explants, each with
approximately 1-cm sides.
The explants are transferred into a 10% hypochlorite bleach solution for 5
minutes, gently agitating once or twice during this time. They are then washed
free of bleach by immersing them in 4 successive beakers of sterile distilled
water, leaving them for 2–3 minutes in each.
Three explants are placed on each petri dish of growth medium, with the
upper epidermis pressed gently against the surface of the agar to make
The petri dishes are sealed with plastic film to prevent moisture loss, and
incubated at 25°C in 16 hrs is light/8 hrs dark.
Step 3: Assessment of Tissue Culture Development
The explants are incubated for 4–6 weeks, and inspected at weekly or
intervals. The growth of obvious bacterial or fungal colonies indicates
and data from such cultures are obviously suspect. The development of
dark brown tissue cultures can also be a consequence of contamination.
The media used in the demonstration are designed to show the effects of
auxin, cytokinin, sucrose, and mineral salts on development. The media were
based on the well-known Murashige and Skoog inorganic media.
After about 8 weeks on each medium typical results appear. To summarize,
multiple adventitious buds form on the control medium, leading to many
shoots on the upper surface where the leaf is not in contact with the
Absence of sucrose inhibits this production. Shoot production is also
on the low sucrose concentration, but comparable with the control at
At zero and low levels of cytokinin, callus forms where the leaf surface
in contact with the medium, while at high levels, shoot formation is
At zero and low levels of auxins there is a stimulus to shoot formation, but
at high concentrations, large numbers of roots are formed.
At low and zero levels of MS salts, there is no growth at all.
These very obvious variations demonstrate the importance of a carbon and
inorganic salt source for plant growth, as well as the effect of the
ratio on the control of plant development.