HISTOLOGY OF SOMATIC EMBRYOGENESIS IN PANICUM MAXIMUM (GUINEA GRASS)

Immature embryos of Panicum maximum Jacq. (Guinea grass) were cultured on Murashige and Skoog's medium containing 2,4-dichlorophenoxyacetic acid (5 mg/l) and coconut milk (5%). The epidermal and subepidermal cells ofthe scutellum at the coleorhizal end either formed somatic embryos directly or gave rise to embryogenic callus at several loci. Somatic embryos were formed later also at the periphery of these embryogenic calli. The embryoids originated from single, densely staining, nonvacuolated and starch-containing cells with thickened walls and developed through characteristic stages of grass embryogeny. SOMATIC EMBRYOS can now be induced in tissue cultures of all major cereal and grass species (Vasil, 1982, 1983, 1985). The determination ofthe origin ofthe somatic embryos-whether from single cells or from groups of cells-is considered important in the isolation of mutants and nonchimeral plants and their use in plant improvement. In a previous study we have reported somatic embryogenesis and plant regeneration from cultured immature embryos and inflorescences of Panicum maximum (Lu and Vasil, 1982). In this report we describe the histology of the formation of embryogenic callus and somatic embryos from cultured immature zygotic embryos of P. maximum. MATERIALS AND METHODS-Immature seeds of Panicum maximum Jacq. 199 (an apomictic selection) were collected from field grown plants. The seeds were surface sterilized and immature embryos (0.5-1.0 mm long) were excised and cultured on Murashige and Skoog's ( 1962) medium supplemented with 2,4-dichlorophenoxyacetic acid (5 mg/l) and coconut milk (5%) as previously described (Lu and Vasil, 1 982). Freshly dissected immature embryos were fixed in formalin-acetic-alcohol for comparison with the cultured embryos. Six to 10 cultured embryos and proliferating tissues were fixed daily for 15 days after culture initiation. Thereafter, fixations were made every 3-4 days until 32 days after initial culture. Fixed tissues ' Received for publication 7 February 1985revision accepted 16 July 1985. We thank Dr. Henry C. Aldrich for use of the Biological Ultrastructure Laboratory, and Dr. Rex L. Smith for providing the experimental plants used in this study. The assistance of Dr. Lewis Berner in the use of his Zeiss Photomicroscope is gratefully acknowledged. Florida Agriculture Experiment Station Journal Series No. 5998. were dehydrated in a tertiary-butanol series and embedded in paraplast. Serial sections were cut at 8-10 ,um and stained with safranin-fast green (Johansen, 1940). Cultured embryos, proliferating calli, and somatic embryos at different stages of development were fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) at room temperature and processed for scanning electron microscopy (SEM) as described by Vasil and Vasil (1984). The tissues were examined and photographed in a Hitachi S-450 scanning electron microscope. The results described are from the examination of embryos cultured during two summer seasons. RESULTS-At the time of culture the embryos showed a well-differentiated shoot-root axis and were fully organized (Fig. 6). Nevertheless, these young embryos were physiologically immature and lacked characteristic storage reserves in the scutellum. The procambium was evident as a strand of elongated cells located centrally in the embryo axis, and it extended also from the scutellar node into the upper half of the scutellum. The scutellum had a single layer of epidermis over its entire surface. Elongation of the embryo and divisions in cells adjacent to the procambium around the scutellar node could be observed 2 days after culture initiation. By this time the scutellar parenchyma cells below the nodal area had enlarged and the abaxial epidermal cells of the scutellum had elongated (Fig. 1, 7). Cell divisions started in the scutellum near the procambium and extended outward to cells of the scutellar epidermis. Except in isolated areas, there was extensive enlargement and dissociation of the scutellar parenchyma at the radicle end. This resulted in the separation of