Demonstration of Tissue Culture for Teaching

Introduction
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 flower— although the success of any one of these varies between species. It is essential that the surface of the explant is sterilized to remove all microbial contamination. Plant cell division is slow compared to the growth of bacteria and fungi, and even minor contaminants will easily overgrow the plant tissue culture. The explant is then incubated on a sterile nutrient medium to initiate the tissue culture. The composition of the growth medium is designed to both sustain the plant cells, encourage cell division, and control development of either an 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 tissue culture is initiated, and how it subsequently develops. The explant should initially form a callus, from which it is possible to generate multiple embryos and then shoots, forming the basis for plant regeneration, and thus the technology of micropropagation. The first stage of tissue culture initiation is vital for information on what combination of media components will produce a friable, fast-growing callus, or a green chlorophyllous callus, or embryo, root, or shoot formation.

There is at present no way to predict the exact growth medium, and growth protocol, to generate a particular type of callus. These characteristics have to be determined through a carefully designed and observed experiment for each new plant species, and frequently also for each new variety of the species that is taken into tissue culture. The basis of the experiment will be media and protocols that give the desired effect in other plant species, and experience.

The Demonstration
The strategy for designing a medium to initiate tissue culture, showing how growth regulators and other factors modulate development, can be demonstrated using the African violet, a popular house plant. Leaf sections are the source of explants. This demonstration is regularly carried out by a student class, and produces reliable results. Sterile supplies are provided from central facilities, and provision of sterile working areas (for example, in laminar flow hoods) is an advantage, although cultures can be initiated in an open laboratory with careful aseptic technique. The standard precautions used during any laboratory work involving chemicals or microbes should be adopted. If you are in any doubt about safety hazards associated with this demonstration, you should consult your local safety advisor.

Step 1: Selection of the Leaves
Leaves are cut from healthy plants, leaving a short length of petiole attached. They should be selected to each yield several explants of leaf squares, with approximately 1-cm sides. The youngest and oldest leaves should be avoided.
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 good contact.

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 biweekly intervals. The growth of obvious bacterial or fungal colonies indicates contamination, 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.

Typical Results
After about 8 weeks on each medium typical results appear. To summarize, multiple adventitious buds form on the control medium, leading to many small shoots on the upper surface where the leaf is not in contact with the medium.

Absence of sucrose inhibits this production. Shoot production is also limited on the low sucrose concentration, but comparable with the control at high sucrose.

At zero and low levels of cytokinin, callus forms where the leaf surface is in contact with the medium, while at high levels, shoot formation is stimulated.

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 auxin:cytokinin ratio on the control of plant development.