@Article{biocell.2008.32.033,
AUTHOR = {LUIS A. MROGINSKI*, PEDRO A. SANSBERRO, ADRIANA M. SCOCCHI, CLAUDIA LUNA, HEBE Y. REY},
TITLE = {A cryopreservation protocol for immature zygotic embryos of species of <i>Ilex</i> (Aquifoliaceae)},
JOURNAL = {BIOCELL},
VOLUME = {32},
YEAR = {2008},
NUMBER = {1},
PAGES = {33--39},
URL = {http://www.techscience.com/biocell/v32n1/37731},
ISSN = {1667-5746},
ABSTRACT = {Tropical <i>Ilex</i> species have recalcitrant seeds. This work describes experiments demonstrating the feasibility of long-term conservation of <i>Ilex brasiliensis, I. brevicuspis, I. dumosa, I. intergerrima, I. paraguariensis, I. pseudoboxus, I. taubertiana</i>, and <i>I. theezans</i> 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± 2ºC on solidified (0.8% agar) 1/4MS medium, [consisting of quarterstrength 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<sup>-1</sup> 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<sup>-2</sup>.s<sup>-1</sup>)/ 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.},
DOI = {10.32604/biocell.2008.32.033}
}