**3. Cryopreservation methods**

The latest results from the field of low temperature biology suggest that the main factor influencing the success of the cryopreservation method is the maintenance of a glassy state in plant samples and the avoidance of ice nucleation. The danger of ice nucleation and subsequent ice crystallization leading to frost damage during cooling and rewarming of samples is considered as a critical point of cryopreservation. That is the reason why many of the new progressive methods use and involve a glassy state in plant material intended for cryopreservation. Knowledge of the glass transition temperature is useful not only for improving methods involving glassy state in plant shoots tips. It also provides information essential for the long-term storage of shoot tips.

This biotechnology is based on the induction of the vitrification status – glass induction by dehydration, addition of cryoprotectants and a very fast decrease in temperature. Vitrification can be achieved in a number of ways (Sakai & Engelmann, 2007) but they usually all have the results of increased solute concentration to a critical viscosity. Low water content minimizes the ice crystallization that is potentially dangerous for plant cells and increases the temperature of glass transition. Supposing that the change of water status in the certain range is not limiting for plant regeneration. Plant Vitrification Solutions (PVS) marked with numbers according to the specific mixture of basic cryoprotectants and their concentrations are usually used for osmotic dehydration. Another cryopreservation method used, is based on desiccation in the air-flow cabinet. It is defined with the flow rate, temperature and humidity or on desiccation over various saturated salt solutions with steady-state activity of water.

for seeding plants for further growing. The vegetatively propagated plant germplasm is endangered by abiotic and biotic factors in the field conditions. Although the production area of many vegetatively propagated plants has been decreasing, many local cultivars and varieties remain. In the presence of decreasing cultivar variability in production areas, diminishing of old orchards, as well as appearance of diseases close to field collection areas, the question of safely maintaining the broad genetic potential of fruit

Two safe methods ensure vegetatively propagated plant germplasm maintenance with a low risk of loss: slow-growth *in vitro* culture and the cryopreservation methods. Advantages of *in vitro* collection are aseptic and stable conditions of the cultivation and availability of the material during the year. A disadvantage is the necessity of sequential plant multiplication. Advantages of cryo-collection are low costs for its long-term maintenance and material stability. Disadvantages are a longer time for the plant to recover from stored material and a rather high input costs of the cryopreservation procedure. The best way how to maintain germplasm is the combination of both methods. The base collection should be maintained by *in vitro* collection that provides the material in case of requirements. Core collection of the most valuable material, should be backedup by cryo-collection for long-term storage, and plants are recovered just in case the genotype is lost from the base collection. For that reason, important vegetatively propagated plant collections have started to introduce accessions to slow-growth *in vitro* cultures and simultaneously in cryo-collection in liquid nitrogen (Gonzalez-Arnao *et al*.,

The latest results from the field of low temperature biology suggest that the main factor influencing the success of the cryopreservation method is the maintenance of a glassy state in plant samples and the avoidance of ice nucleation. The danger of ice nucleation and subsequent ice crystallization leading to frost damage during cooling and rewarming of samples is considered as a critical point of cryopreservation. That is the reason why many of the new progressive methods use and involve a glassy state in plant material intended for cryopreservation. Knowledge of the glass transition temperature is useful not only for improving methods involving glassy state in plant shoots tips. It also provides information

This biotechnology is based on the induction of the vitrification status – glass induction by dehydration, addition of cryoprotectants and a very fast decrease in temperature. Vitrification can be achieved in a number of ways (Sakai & Engelmann, 2007) but they usually all have the results of increased solute concentration to a critical viscosity. Low water content minimizes the ice crystallization that is potentially dangerous for plant cells and increases the temperature of glass transition. Supposing that the change of water status in the certain range is not limiting for plant regeneration. Plant Vitrification Solutions (PVS) marked with numbers according to the specific mixture of basic cryoprotectants and their concentrations are usually used for osmotic dehydration. Another cryopreservation method used, is based on desiccation in the air-flow cabinet. It is defined with the flow rate, temperature and humidity or on desiccation over various saturated salt solutions with

trees is arising.

2008; Keller *et al*., 2008; Kim *et al*., 2006).

essential for the long-term storage of shoot tips.

**3. Cryopreservation methods** 

steady-state activity of water.
