|Authors: ||A. Taglienti, M. Barba|
|Keywords: ||ex situ conservation, virus eradication, genetic stability, regrowth|
The application of cryogenics to plant material is an innovative technique for preservation and recovery of pathogen-free germplasm.
Cryopreservation allows the long-term (theoretically indefinite) storage of plant material; cryotherapy achieves eradication of plant viruses by cryogenic treatment of shoot tips.
The vitrification technique allowed to develop a cryopreservation protocol involving the following steps: cold acclimation of the plantlets, excision of shoot tips and preculture on high sucrose medium, osmoprotection, dehydration with vitrification solution, rapid cooling by plunging in liquid nitrogen, thawing, unloading of vitrification solution and plating on a suitable regrowth medium.
Cold acclimation of plantlets at 4°C in the dark for 10 days was found to be helpful, as much as 1 or 2 days preculture of shoot tips before cryogenic treatment.
Time and temperature for osmoprotection, vitrification and warming were addressed and optimized; the regrowth medium was also adjusted for shoot vitality.
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