Changes in protein metabolism during the acquisition of tolerance to cryopreservation of carrot somatic embryos

High concentrations of sucrose are often used to cryoprserve regenerable pl ant cell cultures in liquid nitrogen. A 21-h pretreatment of carrot somatic embryos in medium containing 0.4 M sucrose allows 80% of them to germinate after freezing. Substitution of sucrose by polyethylene glycol 6000 led to lower germination rates. However, a high level of freezing tolerance was r estored by addition of 1 mu M abscisic acid in the pretreatment medium. Usi ng these different media, both total water soluble protein, using SDS-PAGE, and boiling-stable protein, using 2-D electrophoresis, were studied in rel ation to acquisition of cryopreservation tolerance. Only boiling-stable pro tein patterns showed some changes: five polypeptides accumulated in 0.4 M s ucrose-pretreated embryos or in embryos pretreated by media containing absc isic acid. This accumulation was not detected with polyethylene glycol 6000 used as sole cryoprotectant. Although over-accumulation of polypeptides wa s highest with media containing ABA, the best germination rates were linked to pretreatment with 0.4 M sucrose. The addition of okadaic acid in 0.4 M sucrose medium led to embryo death after freezing, confirming the existence of a message leading to metabolic changes and acquisition of cryotolerance . Water-soluble proteins obtained from 0.4 M sucrose-pretreated embryos app eared more active than those extracted from control embryos in protecting i n vitro a freeze-labile enzyme. Boiling-stable proteins, corresponding to a part of total proteins, were more active than total proteins. These result s suggest that these polypeptides may be involved in a mechanism of protect ion needed for cell survival during freezing stress. (C) Elsevier, Paris.