Tissue Culture & Cryostorage

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

Fontainia australis in 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 such as this Fontainia australis.

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' - which means the ability for a plant to regenerate from a single cell back into a whole plant. Tissue culture is used to mass produce plants. Most commonly tissue culture is a means of propagating plants without seeds. This technique 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 survive and continues to grow, they can be cut into small piece again (allowing plants to be multiplied) and regrown or transferred to a potting mix (exflasking). Because each plant comes from one original cutting 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!

In our laboratory plants are grown in plastic or glass containers placed in controlled growth riins set at about 24 degrees Celsius, under lights (16 hours on and 8 hours off).

Uses of Tissue Culture

  • Large-scale micro-propagation of plants. Many house and garden plants are micropropagated commercially
  • 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

1. Initiation of explants

For plant material (often referred to scientifically as ‘germplasm’) to be tissue cultured, 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. As such, it is washed in soapy water, then surface sterilised. The sterilant most commonly used is sodium hypochlorite (bleach) with a surfactant (detergent) added. To ensure that all residual sterilant is removed, the material is 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 nutrients.

Tissue culture must be done in a sterile environment - plants are gorwn without roots on a growth medium that supplies all the nutrients they require

Macro and micronutrients and vitamins are included 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. 

2. 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 to use 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, subculturing them, and placing them onto fresh medium. This step allows plant numbers to be increased and is carried out frequently in commercial laboratories.

3. Exflasking

Once explants have grown sufficiently in tissue culture they are then transferred from sterile culture to potting mix, in the process known as exflasking. New shoots can be treated as cuttings and transferred directly to the potting mix, however 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 usually requires careful handling, controlled temperature 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.

These Syzygium australe were propogated using tissue culture and were transferred into potting mix once they had roots and were large enough. Once potted up, they were held in a glasshosue to grow further and become hardy - ready for the outside world!

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

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

Cryopreservation

Cryopreservation is the storage of living material in liquid nitrogen at around -196° C. Although the material is still ‘alive’ all cellular activity has stopped leaving the tissue ‘frozen in time.’ As such, cryopreservation provides the longest storage option available for plant material. Seeds, buds, shoots even pollen can be successfully cryopreserved  

Pros and Cons of cryopreservation

There are a number of benefits of cryopreservation including

  • Its relatively low cost compared to other storage methods
  • It requires no additional maintenance as for material stored via tissue culture or living collections
  • It uses minimal storage space
  • It reduces the potential for genetic mutations or contamination of pest and disease.

However, there are also problems with cryopreservation such as the

  • Requirement for specific liquid nitrogen handling facilities and appropriately trained staff
  • Difficulty in the freezing and thawing process which can damage plant tissue
  • Risk of equipment failure or difficultly in obtained regular liquid nitrogen supplies
  • Hazard associated with using liquid nitrogen

Additionally, each plant species and tissue type has very specific requirements for the way in which it is prepared, frozen and thawed meaning each has to be tested individually. Once this information is known, cryopreservation can be successfully applied to many plants

Methods of storing desiccation-sensitive (recalcitrant) species 

Many rainforest species are desiccation sensitive and are therefore difficult to store under standard storage conditions (15°C, 15% RH). Cryopreservation can often be used as a suitable alternative. 
If seeds are to be frozen, firstly, they need to be dried to remove as much ‘free’ water as allowable. Many seeds are then treated with a cryoprotectant (which is similar to the antifreeze in your car radiator) and other solutions to help prevent damage to the cells during storage or the freezing and thawing process. The seeds are now ready to be frozen, and may be placed directly in liquid nitrogen, or have their temperature reduced slowly in a programmable freezer. 
Cryopreservation of plant tissue is completed in a similar manner as seeds, however the tissue (typically generated via tissue culture) is treated with two different cryoprotectants, firstly at a lower concentration and then a higher concentration. This two-step introduction helps to reduce the ‘shock’ to plant cells associated with the strong sugars and salts present in the cryoprotectant solutions. 

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