155 CryoLetters 27(3), 155-168 (2006)  CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK CRYOPRESERVATION OF PLANT GERMPLASM USING THE ENCAPSULATION-DEHYDRATION TECHNIQUE: REVIEW AND CASE STUDY ON SUGARCANE Maria Teresa Gonzalez-Arnao 1* and Florent Engelmann 2, 3 1. Universidad Veracruzana, Facultad de Ciencias Químicas. Prol. OTE. 6, No. 1009, CP 94340, Apartado Postal 215, Orizaba, Veracruz, México. Email:mtgarnao@ivia.es * To whom correspondence should be addressed 2. Cirad, Station de Roujol, 97170 Petit-Bourg, Guadeloupe, French West Indies (present address). 3. International Plant Genetic Resources Institute (IPGRI), Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy. Abstract Encapsulation-dehydration is a cryopreservation technique based on the technology developed for producing synthetic seeds, i.e. the encapsulation of explants in calcium alginate beads. Encapsulated explants are then precultured in liquid medium with a high sucrose concentration and partially desiccated before freezing. Encapsulating the explants allowsthe subsequent application of very drastic treatments including preculture with high sucrose concentrations and desiccation to low moisture contents which would be higly damaging or lethal to non-encapsulated samples. An encapsulation-dehydration protocol comprises the following steps: pretreatment, encapsulation, preculture, desiccation, freezing and storage, thawing and regrowth. Encapsulation-dehydration has been applied to around 40 different plant species. The optimization of the successive steps of the encapsulation-dehydration protocol is illustrated for sugarcane apices. Keywords: encapsulation; dehydration; cryopreservation; germplasm conservation; vegetatively propagated species. INTRODUCTION Most agricultural crops have orthodox seeds, i.e. which can be dehydrated down to low moisture contents and can thus be stored at low temperature for extended periods (44). However, conservation in seed form is problematic for two main categories of plant species, i.e. vegetatively propagated species and species with recalcitrant or intermediate seeds. Cryopreservation, i.e. the storage of biological material at ultra-low temperature, usually that of liquid nitrogen (-196°C) is the only technique currently available to ensure the safe and cost-efficient long-term conservation of these different types of germplasm (14). At this temperature, all cellular divisions and metabolic processes are stopped or minimised. The plant material can thus be stored without alteration or modification for a theoretically unlimited period of time. Moreover, cultures are stored in a small volume, protected from contamination, and require a very limited maintenance. Apices represent the material of choice for cryopreservation of germplasm of vegetatively propagated species. At first, apices were cryopreserved using classical procedures including