SUMMARYA number of fixation techniques were used on cubes of dry cotyledonary tissue of lettuce seed to develop a satisfactory technique for the anhydrous fixation of dry seed material for electron microscopy. Fixation with formaldehyde or acrolein vapours in combination with Spurr's resin embedding produced satisfactory sections, while protocols involving the use of osmium tetroxide vapours resulted in brittle specimens that were difficult to section. The use of glycerol as a vehicle for anhydrous aldehyde fixation, followed by subsequent aqueous processing, introduced artifacts in membrane uitrastructure. When model phospholipid systems, known to exist in the hexagonal phase, were prepared for electron microscopy and compared with anhydrously fixed cotyiedonary tissue, the results suggested that dry seed membranes exist in a lamellar phase. It is proposed that leakage from imbibing seeds may be the result of physical/molecular changes in the membranes rather than of a phase change from the hexagonal to the iamellar state.Key words: Lactuca sativa, anhydrous seed fixation, uitrastructure, aldehyde vapour, osmium tetroxide, hexagonal-lamellar membranes, imbibitional leakage.
INTRODL'CTIONWater uptake by the imbibing seed is characterized by a stage of rapid water uptake, followed by a slower rate of uptake until full hydration is achieved (Simon & Raja Harun, 1972;Leopold, 1980; Murphy & Noland, 1982). Although the leakage of solutes from imbibing seed tissues was initially explained as the result of a phase change in membrane phospholipids from a hexagonal to bilayer state (Simon, 1974), this proposal has not been supported by more recent studies (McKersie & Stinson, 1980;Seewaldt et aL, 1981;Leopold & Vertucci, 1986). It had been suggested that peroxidative changes in seed membranes in dry storage could exert a profound effect on the membrane biJayer at imbibition (Villiers, 1973(Villiers, , 1980Simon 1974). This study reports on attempts to devise a satisfactory method for the electron microscope visualization of cytoplasmic organization and, more especially, the membrane structure of dr>^ seeds. Although there have been many ultrastructural studies on dry seeds in the past (Perner, 1965;Thomson, 1979;Opik, 1980Opik, , 1985Yatsu, 1983), these studies were not specifically aimed at the phase structure of membranes. Furthermore, a recent study (Crowe, Hoekstra & Crowe, 1989) has again suggested that membrane phase transitions are responsible for imbibitional damage in dry pollen. Thus the development of an optimal technique for examining membrane uitrastructure in dr\' seeds is particularly pertinent.
MATERIALS AND METHODS
Plant materialSeeds of lettuce {Lactuca sativa L. cv. Great Lakes) were purchased from a local supplier. Transverse slices of cotyledonary tissue were made with singleedged shaving blades (Schick) which were then cut into cubes of predonninantly uniform tissue of less than 0-5
Aqueous fixationTissue cubes were placed in 5 "" phosphate-buffered gJutaraldehyde (pH 7-4) for 30 min and brief...