ULTRASTRUCTURE OF MAIZE ENDOSPERM SUSPENSION CULTURES

Suspension cultures derived from developing maize (Zea mays L.) endosperm were examined by electron microscopy, after both glutaraldehyde-OsO4 and KMnO4 fixation, and compared with intact endosperm. Tissue clumps consisted of interconnected cell clusters without any organization of the different cell types. The cultures were comprised of cells with dense cytoplasm and small vacuoles, large vacuolate cells, and cells in which storage products (starch, protein bodies, or lipid) accumulated. The endomembrane system of cultured cells was more highly developed than that of cells of the intact endosperm. In particular, arrays of smooth endoplasmic reticulum were seen only in the cultured cells. An abundance of endoplasmic reticulum, dictyosomes, and ribosomes is consistent with the recently reported extracellular secretion of enzymes by these cultures. Cell wall ingrowths, a characteristic of basal endosperm transfer cells, were observed occasionally in cultured cells, but cells with ingrowths had no histological organization. Some of the observed features may have resulted from perturbation of normal cellular events caused by the conditions of in vitro growth. These cultures are a useful tool for studying cellular mechanisms of protein secretion and storage product accumulation in developing maize endosperm. ENDOSPERM CULTURES of maize (Zea mays L.) have been used as a model system to study biochemical events in developing seeds (Shannon, 1982). Cultures derived from endosperms 10 days after pollination have been maintained on agar medium or in liquid suspension for several years. Unlike many plant tissue cultures consisting of undifferentiated cells, maize endosperm cultures maintain their developing endosperm phenotype, and accumulate starch (Chu and Shannon, 1975), zein (Shimamoto, Ackermann, and Dierks-Ventling, 1983), and anthocyanins (Racchi, 1985). Like other plant cells cultured in vitro, endosperm cultures secrete large quantities of acid hydrolases into the medium (Miernyk, 1987). The extracellular secretion of hydrolases, along with the accumulation of zein in the tissue but not in the culture medium, suggests that differential processing of secretory proteins takes place in the tissue. This study was undertaken to correlate the ultrastructure of cultured cells with protein secretion, and to understand more thoroughly the extent of similarity between these cultured endosperm cells and the intact endosperm. MATERIALS AND METHODS-Plant material-A suspension culture derived from maize endosperm (inbred A636) was obtained from ' Received for publication 15 April 1986; revision accepted 6 April 1987. Dr. C. D. Boyer (Pennsylvania State University). Cultures were transferred every 21 days into medium containing inorganic salts (Linsmaier and Skoog, 1965; obtained from GIBCO, Grand Island, NY), 30 g/l sucrose, 2 g/l L-asparagine, and 0.4 mg/l thiamine HCI, adjusted to pH 5.6 (Shannon, 1982). Cultures were incubated on a shaker in the dark at 29 C. At the midpoint of the growth cycle, when growth was most rapid, tissue clumps about 1 mm3 were selected for fixation. Plants of inbred A636 were grown in a greenhouse and hand pollinated. Small slices (< 1 mm3) oftissue containing aleurone, subaleurone, and a portion of the internal endosperm were obtained with a razor blade from the middorsal region of grains sampled 15 days after pollination. Microscopy-Tissue was fixed in 2% (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 4 hr at 25 C, rinsed in 4 changes of buffer over a period of 1 hr, and postfixed in 1% (w/v) OS04 in the same buffer for 2 hr. The tissue was dehydrated in an acetone series and embedded in Effa-poxy2 resin (Ernest F. Fullam, Inc., Schenectady, NY) by slowly increasing the ratio of resin mixture to dry acetone. After infiltration for 24 hr on a rotating plat2 Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable.