Cryopreservation of immature maize embryos after freeze-hardening in the ear and in vitro

We have studied cryopreservation of immature maize embyros. Common cyroprotective treatments, i.e., pretreatment with dimethysulfoxide and glycerol, followed by slow-freezing, were not successful. By freeze-hardening, we obtained a high percentage of embryos which survived cryopreservation. The freeze-hardening treatments consisted of either leaving the embryos in the kernels of a detached ear or culturing excised embryos in vitro on solid media containing high osmoticum. In both freeze-hardening treatments, success was only achieved when the embryo water content had decreased below a critical level (71%). Other factors were also important. When embryos were left in the kernels of a detached ear, they likely entered the desiccation-tolerant phase rapidly as is shown by the sharp increase of dry weight and of asparagine/aspartic acid content. During culture in vitro on high osmoticum, the embryos showed the usual physiological response to water stress, i.e. proline accumulation. Possibly, water stress induced a metabolism which made the embryos resistant to freezing damages. Freeze-hardening by culture in vitro on high osmoticum might be a suitable treatment for the cryopreservation of in vitro material and germplasm storage.