Cryopreservation (preservation in liquid nitrogen, -196°C) of plant germplasm

       The term ‘cryopreservation’ (cryogenic preservation) refers to the storage of cells, tissues and organs at the ultra-low temperature of liquid nitrogen (-196°C). At this temperature, the vegetative cells enters in a state of “absolute quiescence”, as all the physical and biochemical reactions are practically halted; in this particular condition, conservation time becomes unlimited. Application of cryogenics to the conservation of plant material, proposed for the first time in the year 1968 for the maintenance of cell cultures, is today a reality for the conservation of differentiated organs and tissues as well. When the cells are led to this extreme ultra-cold condition following appropriate “preparatory” procedures, their viability is preserved and, when brought back to standard culture conditions, they can recover full functionality. Among the main advantages of cryopreservation, there are the possibility to put in conservation a wide range of plant organs and tissues (shoot tips from in vitro culture, seeds and embryonic axes, somatic embryos, bulbils, buds collected in the field, pollens), the limited space necessary for the conservation (a medium-size dewar for liquid nitrogen contains 5,000-10,000 explants), the low conservation cost (practically, the cost necessary for the cryobank control and for the provision of liquid nitrogen, a liquid gas cheap and easy to find), the maintenance of specimens in absolute genetic and sanitary security.


  Furthermore, in liquid nitrogen it is possible to preserve cell and hairy root cultures producing secondary metabolites of industrial and pharmacological interest, as well as callus cultures, embryogenic and/or genetically transformed. Recently, the cryogenic tecnology showed its effectiveness also for the recovery of virus-, phytoplasm- and bacterium-free plants (cryotherapy). Since more than a decade, a research group of the CNR-IVALSA/Istituto per la Valorizzazione del Legno e delle Specie Arboree (Trees and Timber Institute), coordinated by Dr. Maurizio Lambardi, studies the application of the cryogenic technology to the conservation of woody plant germplasm (fruits, timber and ornamental species). Effective procedures have been developed for the cryopreservation of shoot tips from white poplar, pear, plum, kaki and olive, seeds of Citrus and Pistacia spp., embryogenic callus of olive, horsechestnut and ash. The vitrification solution PVS2 is largely used with both naked and encapsulated explants (synthetic seeds). An innovative procedure, based on the cryopreservation of dormant buds which, after thawing, are used for chip-budding onto rootstocks, has been recently applied to the conservation of ancient apple germplasm. Important information on the micro-morphological characteristics of explants recovered from cryopreservation are obtained by means of histo-anatomical observations.

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