Lambardi and De Carlo - Techniques and applications of cryogenics to the conservation and recovery of pathogen-free plant germplasm

Maurizio Lambardi* e Anna De Carlo

nsegna_

Key words: Ex situ conservation, Cryobiology, Cryopreservation, Genetic resources, Vitrification.

nsegna_

Abstract

nsegna_

Cryopreservation (cryogenic preservation) refers to the conservation in liquid nitrogen of cells, tissues and organs from in vitro culture (shoot tips, embryogenic callus, somatic embryos), as well as from in vivo collected material (seeds, embryonic axes and dormant buds). The technique opens important prospects to the safeguard of plant germplasm, allowing the storage of plant material at low cost, for unlimited time and in absolute sanitary and genetic safety, as at the temperature of liquid nitrogen (-196°C) all the biochemical and physical cell processes are completely arrested. For long time, the slow-cooling technique has been the only approach to plant cryopreservation. Here, cell vitrification (i.e., the solidification of liquids in a glassy state which prevents the formation of ice crystals) is achieved by cryodehydration, i.e., slowly cooling (-1°C min-1) the explants down to -40°C and then plunging them into liquid nitrogen. In the last 20 years, new techniques have been developed, such as the treatment with vitrification solutions, the droplet-method, the encapsulation-dehydration, the encapsulation-vitrification and the seed dehydration. All these techniques are based on the osmotic or evaporative dehydration of explants which can be afterwards directly immersed in liquid nitrogen, thus producing a marked simplification of cryogenic procedures. The development of such new techniques paved the way to the optimization of effective cryopreservation protocols for numerous horticultural species (vegetables, ornamentals and fruits), mainly by the treatment of shoot tips with PVS2 (a highly concentrated mixture of three cryoprotectants: glycerol, DMSO and ethylene glycol) or by the dehydration in air flow or on silica gel of alginate- encapsulated explants (“synthetic seeds”). Moreover, cryopreservation of dormant buds has also been very successful for the conservation of fruit species reproduced by budding propagation. The procedure is based on winter collection of scions from which uni-nodal sections are cut, desiccated, slow cooled (-1°C h-1) down to -30°C, stored in liquid nitrogen, thawed, re-hydrated and chip budded onto clonal rootstocks. Recently, cryotherapy (i.e., the use of liquid nitrogen to recover pathogen-free plants) showed to be a promising approach to the eradication of viruses, phytoplasma and bacteria from infected material. The technique allows treatment of large numbers of samples and results in a high frequency of pathogen eradication, being of strategic importance to achieve the simultaneous production and long-term storage of pathogen-free plant genetic resources. The EU shows great attention and interest to this research area, promoting the onset in 2006 of the COST Action 871 (“CryoPlanet, Cryopreservation of Crop Species in Europe”) which gathers scientists from 18 European countries. The ultimate profit of the technology is the establishment of cryogenic repositories (“cryobanks”) for the safeguard of plant biodiversity and promising examples are already existing in the world. The organization of a shoot-tip cryobank for the conservation of clonal germplasm is described.

nsegna_

 Download (members only)

Download full article

Lambardi-De Carlo.pdf