An ultrastructural and cytochemical study of the wall-membrane apparatus of transfer cells using freeze-substitution

A freeze-substitution technique is described which enables the ultrastructure of certain types of plant transfer cells to be preserved with minimal ice crystal damage. The ultrastructure of transfer cells from Funaria, Lonicera, and Senecio after freeze-substitution has been compared with that of glutaraldehyde-osmium fixed material. The irregular clear zone between wall and plasma membrane, present in conventional preparations, is absent in freeze-substituted tissue. It is proposed that this interfacial zone is an artefact caused by expansion of wall ingrowth material during conventional fixation procedures. In transfer cells with a complex wall labyrinth the swelling of wall material severely disrupts the true structure of the wall-membrane apparatus and results in a large decrease in the surface to volume ratio of the protoplast. These findings are supported in the case of Funaria by a freezefracture study. The reactivity of the plasma-membrane to the PTA/chromic acid stain is enhanced in freeze-substituted material. Use of the Thiéry silver proteinate reagent in conjunction with freeze-substitution has revealed marked differences between the wall ingrowths of Funaria sporophyte haustorium transfer cells and those of Lonicera nectary trichomes.