Cryopreservation of Ilex Immature Zygotic Embryos

Tropical Ilex species have recalcitrant seeds. This chapter describes protocols for long-term conservation of Ilex brasiliensis , I. brevicuspis , I. dumosa , I. microdonta , I. integerrima , I. paraguariensis , I. pseudoboxus , I. taubertiana , and I. theezans through cryopreservation of zygotic rudimentary embryos at the heart developmental stage. The embryos are aseptically removed from the seeds and precultured (7 days) in the dark at 27  �  2�C on solidified quarter-strength Murashige and Skoog medium with 3% sucrose and 0.1 mg/L zeatin. The embryos are then encapsulated in 3% calcium alginate beads and pretreated at 24-h intervals in liquid medium supplemented with progressively increasing sucrose concentrations (0.5, 0.75, and 1 M). The beads are dehydrated for 5 h with silica gel to 25% water content (fresh weight basis) and then placed in sterile 5-mL cryovials. Then the beads are either plunged rapidly in liquid nitrogen where they are kept for 1 h (rapid cooling), or cooled at 1�C/min to −30�C and then immersed in liquid nitrogen for 1 h (slow cooling). After cryopreservation, the beads are rewarmed by immersion of the cryovials for 1 min in a water bath at 30�C. Finally, the beads are transferred onto culture medium (1/4MS, 3% sucrose, and 0.1 mg/L zeatin, solidified with 0.8% agar) and incubated in a growth room at 27  �  2�C under a 14-h light (116 μmol/m² /s) and 10-h dark photoperiod. Maximum recovery percentages between 15 and 83% (depending on the species and the treatment) were obtained with the cryopreserved embryos.