325 CryoLetters 23, 325-332 (2002)  CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK CRYOPRESERVATION OF Citrus madurensis EMBRYONIC AXES BY ENCAPSULATION-DEHYDRATION Eun Gi Cho 1, 3* , Y.L. Hor 2 , Haeng Hoon Kim 3 , V. Ramanatha Rao 1 and Florent Engelmann 4, 5 1 International Plant Genetic Resources Institute (IPGRI), Regional Office for Asia, Pacific and Oceania, P.O. Box 236, UPM Post Office, 43400 Serdang, Selangor Darul Ehsan, Malaysia. 2 Department of Crop Science, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia. 3 National Institute of Agriculture and Science Technology, RDA, Suwon 441-707, Republic of Korea. (permanent address for E.G.C.) (email for E.G.C.: e.cho@rda.go.kr). 4 IPGRI, Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy. 5 Institut de recherche pour le développement (IRD), BP 5045, 34032 Montpellier Cedex 01, France. (current address). Abstract In this paper, we demonstrate that C. madurensis embryonic axes can withstand cryopreservation using the encapsulation-dehydration technique. Up to 57.5 % survival was achieved using a standard encapsulation-dehydration protocol, which included pregrowth of encapsulated axes for 16 h in medium containing 0.8 M sucrose + 1 M glycerol, desiccation of beads to around 30 % moisture content (fresh weight basis) followed by rapid freezing. A slightly higher survival percentage (65 %) was obtained using a modified encapsulation- dehydration protocol, which included pretreatment of axes with 2 M glycerol + 0.6 M sucrose for 1 h, concomitantly with their encapsulation in 3 % calcium alginate beads, followed by desiccation of the beads to around 30 % moisture content. Keywords: Citrus madurensis; embryonic axes; cryopreservation; encapsulation-dehydration; pretreatment. INTRODUCTION Various techniques are available for the cryopreservation of plant germplasm, among which is the encapsulation-dehydration technique. This technique, established by Dereuddre et al. (3) using apices of pear in vitro plantlets, is based on the technology developed for the production of synthetic seeds, where somatic embryos are encapsulated in a bead of hydrosoluble gel. The principle of the encapsulation-dehydration technique is that encapsulated explants can be submitted to very drastic treatments (pregrowth with high sugar concentrations followed by extensive desiccation) which would be lethal to non-encapsulated material. Encapsulation-dehydration has been mainly applied to apices from in vitro plantlets of numerous temperate and tropical plant species, as well as to somatic embryos of several