Description

ABSTRACT

Changes in cell ultrastructures at different steps of cryopreservation protocol application under light microscope and transmission electron microscope (TEM) on Chinese yam (Dioscorea polystachya Turcz.) were studied. The specimen preparation for the examination was accomplished through steps of liquid fixation, dehydration, embedding and sectioning on an ultramicrotome followed by staining before observation. Following preculture treatment, an initial stage of vacuoles fragmentation, increasing numbers of amyloplasts containing starch granule and undulated nuclear envelopes were observed. Cells with small vacuoles containing high amount of dark-staining structures were observed after the dehydration treatment. Rapid rewarming of the explants was followed by some living cells with spherical shapes of nuclei and nucleoli, fragmented vacuoles containing electron dense globular and multi-membranous structures, osmiophilic bodies which were encountered along the plasmalemma, as well as some invaginations of the plasmalemma. Unloading treatment following rapid rewarming caused disappearance or inconspicuous appearances of cellular organelles in many cells. Several surviving cells showing disrupted nuclei; others have expanded nuclear envelopes at different sites. The protocol developed for cryopreservation should be simple and include only necessary steps that enable an optimum recovery. Addition of unnecessary steps can impose additional cellular injuries and reduce the quantity and quality (shoot or callus) of survival

Keywords: Cryopreservation, Cell ultrastructure, Chinese yam, TEM, Vacuole fragmentation, Osmiophilic bodies