Cryopreservation for the safeguarding of woody plant biodiversity

The preservation of plant biodiversity combats the risk of "genetic erosion", i.e., the risk that species, forms and plant varieties may become extinct, producing a definitive loss of the genetic variability which they contain. It is estimated that today over 9000 species can be considered endangered in the world. As concerns woody plants, the cultivation of which is characterized by the use of a more and more restricted number of selected cultivars in fruit and timber plant orchards, the establishment of in-field clonal collections (for vegetatively propagated species) and of seed banks (for seed propagated species) are the traditional approaches to ex situ germplasm preservation. However, clonal orchards require vast areas of lands, their management is very expensive, and the system runs the risks arising from attacks of strong pathogens. As regards seed conservation, many woody species have non-orthodox seeds which cannot be preserved this way. Hence, the conservation of germplasm in liquid nitrogen (cryopreservation) could be an important complementary approach to the safeguarding of woody plant biodiversity, assuming that the development of low-input and widely-applicable technology is pursued.
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Cryopreservation is the storage at ultra-low temperature (-196°C, the temperature of liquid nitrogen) of organs and tissues from in vitro culture, such as buds, shoot tips, embryos and cell cultures. New cryogenic techniques are under experimentation, aiming at the direct immersion in liquid nitrogen of plant specimens from tissue culture, without resorting to expensive apparatus for slow cooling and with a considerable simplification of procedures. This technology is based on the induction of explant "vitrification" during a very fast decrease of temperature. "Vitrification" of cells and tissues is the physical process which avoids intracellular ice crystallization during ultra-freezing. As a consequence of this process, plant tissues are protected from damage and remain viable during their long-term storage at -196°C. Using this technique, the Trees and Timber Institute of Florence is carrying out experiments with several fruit and forest species. Considerable results have already been achieved with the cryopreservation of shoot tips of white poplar (Populus alba; Fig. 1) and plum (Prunus domestica; Fig. 2a), embryogenic lines of olive (Olea europaea), horsechestnut (Aesculus hippocastanum) and ash (Fraxinus angustifolia; Fig. 2b), and polyembrionic seeds of Citrus spp. (Fig. 2c). The study is partly supported from the SCRIGNO Project (aim "Characterization of cherry germplasm of Tuscany and Umbria"), and it receives a grant of the Regione Veneto, project "Collection, conservation and exploitation of autochthonous germplasm of pome fruits", in collaboration with Veneto Agricoltura.
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CNR, Trees and Timber Institute, Florence.

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