Cryopreservation in Forestry
Tannis Beardmore

Cryopreservation, the storage of materials at -196^oC, is a useful method for long-term storage of a wide variety of plant germplasm (i.e., buds, seed, seed parts, twigs, tissue cultures, cells, DNA etc.).

1. Cryopreservation can be used to preserve valuable germplasm.

• Many of Canada's native tree species, primarily hardwoods, produce seed which deteriorate very quickly using traditional storage methods (i.e., storing seed in jars at 0 to -20^oC).
• Cryopreservation is a method that can be used to store seed or other types of germplasm in the long-term. This type of work allows for the conservation of these genetic resources, contributing to the maintenance of Canada's forests.
• A very important example of the potential of cryopreservation is the ex situ conservation of Butternut Juglans cinera L. Butternut is being killed throughout its range by a fungus most likely introduced from outside of North America. Cryopreservation will provide a source of disease-free material to replenish and reintroduce butternut to areas devastated by the disease.

• Somatic embryogensis is a cloning technology that is being incorporated into industry.
• Using cryopreservation as a storage tool has provided an indefinite source of tissue culture material that can be used for mass production or artificial seed. Artificial seed technology is still at the development stage.

• It is the study of phenomena at low temperatures (-73^oC and lower).

• Cryopreservation is the freezing materials at -196^oC.

• The common method is to use liquid nitrogen, which is made by the fractional distillation of liquified air.

• Usually methods need to be developed for cryopreserving different types of germplasm.
• Two factors are important for survival at 196^oC: 1) the types of treatments you expose the germplasm to prior to cryopreservation (i.e., seed treatments, exposure of the tissue to cryoprotectants such as sucrose, etc.); and 2) the rate at which you cool the germplasm.

• First, the temperature of the tissue is cooled from room temperature to -40^oC using a liquid nitrogen cooled freezer.
• When the tissue has reached a temperature of -40^oC it is held at that temperature for 2-3 hours and then is placed directly in liquid nitrogen at -196oC.

• The freezing process which occurs during cryopreservation is not completely understood. Generally, water is the main component of living cells and all cellular mechanisms stop as this water is converted to ice.
• Cells which are frozen by the two-step cooling process are believed to first form ice outside of the cell (extra-cellular freezing), and as this occurs the water inside of the cell (intra-cellular water) moves outside of the cell before freezing. This process allows the cell to dehydrate and when warmed the water then moves back into the cell and cellular metabolism is restored.

• Cryogenic temperatures stabilize cells and there are no changes to these cells while they remain at these temperatures.
• Cryogenics methods have been developed and are being developed for the preservation of germplasm from various tree species in Canada.
• Cryogenics are routinely used in forestry in somatic embryogenesis programs.
• Cryogenic treatments are used on every vessel sent into outer space. This treatment creates a denser molecular structure which results in a greater contact surface area, reducing the wear and tear effects of friction, and heat.
• The lowest temperature reached is one millionth of a degree above absolute zero (equivalent to -273^oC), using infrared lasers to reduce the energy of cesium atoms in a vacuum.
• Some unusual conditions occur at cryogenic temperature allowing for superconductivity, superfluidity, large-scale quantum effects.
• Companies in the US will cryogenically freeze human bodies (at an initial cost of $120,000.00).