An Aceto-Carmine Squash Technic for Mature Embryo Sacs

A method is described by which, whole embryo sacs of Nicotiana, Petunia and no doubt of certain other genera can be obtained readily in aceto-carmine ovule squashes. Although application of the technic to megagametogenesis and fertilization stages is stressed in this paper, use of the method allows development to be traced from the archespore up to the second or third division of endosperm nuclei. The success of the technic depends on four phases:-1) fixation in a medium that causes cell and nuclear structures to become pliable, yet rigid enough that their spatial relationships are not greatly distorted in squashing; 2) heat, which apparently increases the cohesion of cytoplasmic and nuclear constituents; 3) maceration to separate the embryo sac from surrounding cells; and 4) the use of a stain that differentiates the various nuclear structures as well as those of the cytoplasm. Staining of the cytoplasm, essential in some embryological investigations, is one advantage of the aceto-carmine squash method over the Feulgen procedure. In contrast to the Feulgen ovule squash method the aceto-carmine technic will probably be most useful in genera having numerous small ovules. Advantages and defects of the aceto-carmine procedure as compared with the paraffin technic are discussed, likewise the possible usefulness of the former in studies of sterility and in certain other special connections.