Cytochemical and Autoradiographic Analyses on the Embryo Suspensor Cells ofPhaseolus Coccineos

Abstract

Specific staining methods (Feulgen, methyl green-pyronin, toluidine blue molybdate, acridine orange), digestion tests (DNase and RNase) and autoradiographic techniques (incorporation of tritiated thymidine, uridine, lysine, tryptophan; DNA detection by means of tritiated actinomycin D) have been used to study the embryo suspensor of Phaseolus coccineus. Most of material studied consisted of developing seeds in which the cotyledons of the embryo occupied one third to one half of the endospermatic cavity. In the typically club-shaped suspensor, both the « handle » portion (which contains cells with low to medium degree of endopolyploidy) and the « knob » portion (which contains polytene chromosome cells) have been analyzed. Indications have been found that the cells with low and medium degree of endopolyploidy undergo extra DNA synthesis (gene amplification) on many heterochromatic chromosome regions during early stages of embryogenesis. Later on, the extra DNA synthesized seems to be released, from the chromosomes into the nucleoplasm and from here into the cytoplasm, as micronucleoli or clusters of micronucleoli. The micronucleoli are formed by a spherical mass of ribonucleoproteins covered by a layer of DNA, sometimes at least, traversed by thin DNA strands. In addition to extrusion into the cytoplasm of micronucleoli, the nuclei of the « handle » portion of the suspensor are active in the extrusion of nuclear material in form of « blebs »: whether these contain only ribonucleoprotein or also bear DNA in a condition different from that of the micronucleoli cannot be decided. At the developmental stages studied, the cells of the « handle » portion of the suspensor do not practically incorporate thymidine and only rarely incorporate lysin (in 2 out of 11 suspensors studied); but they are active in the incorporation of tryptophan and uridine. After 30 minutes of feeding with ³H-uridine, labelling in the suspensor nuclei is restricted to the nucleoli, whilst in the embryo cells (both plumule and cotyledons), the nuclei (chromatin) are heavily labeled, but the nucleoli are practically unlabeled. In the polytene chromosome cells of the suspensor (« knob » portion) the following observations have been made: The phenomena observed in P. coccineus find a striking parallel in the phenomena described in the polytene chromosome cells of Sarcophaga, Hybosciara and, occasionally, in Chironomus.