Tissue culture

Tissue culture is a series of processes used to grow whole plants from very small pieces of plant tissue.

At the Australian PlantBank, tissue culture techniques are regularly used to maintain cultures of flannel flower and waratah cultivars. Tissue culture is also used to produce material for cryopreservation for those species not suited to seedbanking, such as rainforest seeds.

What is tissue culture?

Tissue culture involves taking very small pieces of plant (bud, shoot tips, or other parts) and growing them on special nutrient media in sterile conditions. This method allows the production of many plants from a single shoot - a great advantage when plants are rare – and for species that do not reproduce by seed.

The plants are grown under sterile conditions on liquid or solid medium that contains nutrients and one or more plant growth regulators. The key to tissue culture is 'totipotency' – this is the ability for a plant to regenerate from a single cell back into a whole plant.

Tissue culture is also known as micropropagation.

Just like you might take a cutting from your garden, dip it in root power and pot it up, small pieces of plant tissue are carefully placed in a jelly-like substance called agar and allowed to grow (initiation). If the plant survives and continues to grow, they can be cut into small pieces again (allowing plants to be multiplied) and regrown or transferred to a potting mix (exflasking).

Each plant comes from one original cutting, so they are all identical (clones) and therefore tissue culture can be used to propagate and multiply thousands of the same plant over and over again. This is great when a plant is highly valued, or when researchers require identical plants of the same age, size and even genetic type!

At PlantBank, our tissue culture plants are grown in plastic or glass containers placed in controlled growth rooms set at about 24 degrees Celsius, under lights (16 hours on and 8 hours off).

Uses of Tissue Culture

Large-scale micropropagation of plants. Many house and garden plants are mass produced using tissue culture.
To conserve highly threatened species, particularly when there is a shortage of material available for conservation
To reproduce plants which don’t produce seed or don’t germinate well
To conserve plants not suited to seedbanking e.g. rainforest seeds
To germinate seeds of species which require a mycorrhizal partner in the wild e.g. terrestrial orchids
To enable the transport of plants to other states or countries without transporting pathogens or pests
To prepare material for cryopreservation and to recover it after freezing.

The stages of tissue culture

Initiation of explants

For plant material (often referred to scientifically as ‘germplasm’) to begin tissue culture, the process starts with initiation. Shoots and nodes are collected from a plant’s fresh new growth. Embryos or even groups of cells can also be used.

The plant material or ‘explant’ needs to be as clean as possible at the beginning of the process to limit bacterial and fungal contamination. So the explant is washed in soapy water, then surface sterilised (often using sodium hypochlorite, bleach, with added detergent). The material is then rinsed in sterile water before being cut into small pieces, each containing a single node in the case of shoots.

Rinsing and sectioning of plant material is completed in a sterile environment to reduce the chance of microbial contamination. The material is then placed on an agar-based medium containing sucrose and other macronutrients, micronutrients and vitamins to promote plant growth.

The material is monitored for up to 4 weeks for fungal or bacterial contamination. Any material that shows signs of contamination or has died off is eliminated, while clean, living material goes to the next stage.

Growth and multiplication

Clean explants are placed on a medium containing growth regulators to encourage shoot growth. A growth regulator is a chemical that mimics plants natural hormones and determines how the explants grow.

The relative proportion of cytokinins (growth regulators which encourage shoot multiplication) and auxins (growth regulators with encourage root formation) determines whether shoots or roots are produced. A higher proportion of cytokinins is required at this stage to generate shoots.

Some experimentation can be required at this stage to determine the best combination of growth regulators for a particular species. Once a suitable medium is found to promote plant growth, subculturing is required at regular intervals.

Subculturing involves removing newly generated shoots, dividing them up, and placing them onto fresh medium. This step allows plant numbers to be increased and is carried out frequently in commercial laboratories.

Exflasking

Once explants have grown sufficiently in tissue culture, they are then transferred from sterile culture to potting mix, in a process known as exflasking.

New shoots can be treated as cuttings and transferred directly to the potting mix, but some cuttings require roots to be propagated successfully. If roots are required, the shoots are transferred onto a medium that contains a higher proportion of auxins. Again, some experimentation may be required to determine the best combination of growth regulators to use for a given species.

Explants removed from tissue culture are very delicate and require careful handling, controlled temperatures and high humidity until the tissues have had an opportunity to ‘harden-off’. This is usually in a controlled environment such as a glasshouse. Eventually the explants re-establish or acclimatise to the conditions outside of the tissue culture jar and become fully-functioning plants again.

For more information see Plant Germplasm Conservation Guidelines by the Australian Network of Plant Conservation.