Cryopreservation of Ilex immature zygotic embryos

Abstract

Tropical Ilex species have recalcitrant seeds. This work decribes protocols demonstrating the feasibility of long-term conservation Ilex brasiliensis, I. brevicuspis, I. dumosa, I. microdonta, I. intergerrima, I. paraguariensis, I. pseudoboxus, I. taubertiana and I. theezans through cryopreservation of zygotic rudimentary embryos at the heart developmental stage. The embryos were aseptically removed from the seeds and precultured (7 days) in the dark, at 27± 2°C on solidified (0.8% agar) 1/4MS medium, (consisting of quarter-strength salts and vitamins of Murashige and Skoog [1962] medium) with 3% sucrose and 0.1 mg/l Zeatin. The embryos were 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). Beads were dehydrated for 5 h with silicagel to 25% water content (fresh weight basis) and then placed in sterile 5 ml cryovials. Then the beads were either plunged rapidly in liquid nitrogen were they were kept for 1 h (rapid cooling) or cooled at 1 °C min-1 to -30 °C. Then the beads were immersed in liquid nitrogen for 1 h (slow cooling). The beads were rewarmed by immersion of the cryovials for 1 min in a water bath thermostated at 30 °C. Finally, beads were transferred onto culture medium (1/4MS, 3% sucrose, 0.1 mg/l zeatin, solidified with 0.8 % agar) and incubated in a growth room at 27 ± 2º C under a 14 h light (116 μmol. m-2.s-1)./ 10 h dark photoperiod. Maximum recovery percentages between 15 and 83% (depending on de the species and the treatment) were obtained with the cryopreserved embryos.