IMPROVED ULTRASTRUCTURAL PRESERVATION OF PETUNIA AND BRASSICA OVULES AND EMBRYO SACS BY HIGH-PRESSURE FREEZING AND FREEZE-SUBSTITUTION

In order to improve the ultrastructural preservation of the female gam etophyte of Petunia x hybrida and Brassica napus we tested several cry ofixation techniques and compared the results with those of convention al chemical fixation methods. Ovules fixed with glutaraldehyde and osm ium tetroxide in the presence or absence of potassium fenocyanide show ed poor cell morphological and ultrastructural preservation. In ovules cryo-fixed by plunging into liquid propane, the cell morphology was w ell preserved. However, at the ultrastructural level structure-distort ing ice crystals were detected in all tissues. Due to the large size o f the ovules, cryofixation by plunging in liquid propane is not adequa te for ultrastructural studies. In contrast, P. I hybrida and B. napus ovules cryo-fixed by high pressure freezing showed improved cell morp hological as well as ultrastructural preservation of the embryo sac an d the surrounding integumentary tissues. The contrast of the cellular membranes after freeze substitution with 2% osmium tetroxide and 0.1% uranyl acetate in dry acetone was high. At the ultrastructural level, the most prominent improvements were: straight plasma membranes which were appressed to the cell walls; turgid appearing organelles with smo oth surface contours; minimal extraction of cytoplasmic and extracellu lar substances. In contrast to the chemically fixed ovules, in high pr essure frozen ovules numerous microtubules and multivesicular bodies c ould be distinguished.