A cryopreservation protocol for immature zygotic embryos of species of Ilex (aquifoliaceae)
Fecha
2008-04Autor
Mroginski, Luis Amado
Sansberro, Pedro Alfonso
Scocchi, Adriana M.
Luna, Claudia Verónica
Rey, Hebe Yolanda
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Tropical Ilex species have recalcitrant seeds. This work describes experiments demonstrating
the feasibility of long-term conservation of Ilex brasiliensis, I. brevicuspis, I. dumosa, I. intergerrima, I.
paraguariensis, I. pseudoboxus, I. taubertiana, and I. theezans through cryopreservation of zygotic rudimen-
tary embryos at the heart developmental stage. The embryos were aseptically removed from the seeds and
precultured (7 days) in the dark, at 27± 2oC 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 1oC min-1 to -30oC. 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
30oC. 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 ± 2oC 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.
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