One of the youngest collections at the PAN Botanical Garden - CZRB in Powsin is the cryogenic Gene Bank of historic apple varieties (Malus domestica Borkh.). Its establishment is linked to a development project "Application of cryogenic methods to establish a gene bank of historical apple varieties". conducted in 2009-2012 with funding from the National Research and Development Center. In our country, this was the pioneering project of its kind for apple trees.

The purpose of the work undertaken in its creation was to safeguard valuable apple tree varieties, no longer found in commercial orchards and gradually receding from amateur cultivation. The cryogenic collection of apple trees complements the protection of genetic resources, which for many years has been the species' pomological orchard Botanical Garden. This allows to increase the level of protection of the collected varieties. This is possible due to the fact that the samples collected at the Gene Bank are completely isolated from the adverse effects of weather conditions, pest and pathogen pressure and possible natural disasters.

The main steps in the process of including apple varieties in the bank include:

Collection of plant material - The object of our interest is resting buds (so-called sleeping buds) extracted in winter, from one-year-old shoots.

Fragmentation - The shoots are cut into sections containing one bud.

Drainage - the treatment necessary for tissue-safe freezing is to deprive them of excess moisture. Despite harvesting the buds when they were at their lowest mid-year hydration (about 50%), they still had too much moisture. At negative temperatures (-4oC) dehydration to 30%, which is the optimum moisture content for apple trees, is carried out.

Placing single-bud fragments in cryofilaments and describing them (fig.1)

Freezing - is carried out gradually at a rate of 0.1oC/min, from a temperature of about 0oC to -40oC. This rate and range allow safe freezing of tissues minimizing the possibility of tissue damage caused by ice crystals (figs. 2, 3)

Inclusion in the bank - Tubes containing 2-3 buds are transferred to a tank cooled with liquid nitrogen. As a target, the temperature of its vapor (about -160°C) is used. For each variety, at least 50 sleeping buds are protected in this way. Under the influence of such a low temperature, tissue metabolism stops, completely reducing the loss of vitality associated with the aging process. As a result, plant material can be stored long-term without loss or alteration of genetic information. In addition, the size of the collection carried out in this way is significantly reduced, which is achieved by placing sample boxes in multi-level racks (figs. 4, 5)

Vitality assessment - It is undertaken in the summer (mid-July) in the year of harvesting buds. After thawing the samples in a water bath (15 min, 35oC), the buds are subjected to hydration in moist peat for a week (temperature 4oC). The buds are then caulked (grafted) onto rootstocks, and their regeneration is observed until the end of the current and the beginning of the new season (Fig. 6). The multi-year average of regeneration for varieties is more than 66%.

Thanks to the cryogenic collection of apple trees, we conduct an alternative form of protection for this species and have an "iron reserve" of its valuable varieties.

Current composition of the collection