MORPHOLOGY AND ULTRASTRUCTURE OF EMBRYOGENIC CELL SUSPENSION CULTURES OF PANICUM MAXIMUM (GUINEA GRASS) AND PENNISETUM PURPUREUM (NAPIER GRASS)

Morphological and ultrastructural changes during the growth of embryogenic cell suspension cultures of Panicum maximum and Pennisetum purpureum have been studied. The suspensions consist almost entirely of cell aggregates of 50-75 embryogenic cells. The cell aggregates vary in size from 90-400 ,m in P. maximum and from 70-340 ,um in P. purpureum. Following the period of exponential growth starch grains gradually disappear and vacuolation increases. Ten to 16 days after subculture, P. maximum cells enlarge and separate from each other, and organized embryo-like structures appear. Ultrastructural studies show that the cell aggregates are made up of discrete, individual groups of 2-6 cells each. Each cell group appears to arise from a single cell and breaks away from the 'mother group' as cell divisions continue. The embryogenic cells are small (20 ,um), isodiametric with many starch grains and contain a large nucleus with a prominent nucleolus. Extensive profiles of endoplasmic reticulum, many small peripheral vacuoles, and several amyloplasts are present. Plasmodesmatal connections exist only between cells within a cell group but not between cells of different cell groups in the large cell aggregates. RECENT ADVANCES NOW make it possible to regenerate plants, via the formation of somatic embryos, from tissue cultures of a wide variety of cereal and grass species (Vasil, 1982, 1985; Dale, 1983). Embryogenic callus cultures have been used to develop lines of embryogenic cell suspension cultures in Panicum maximum (Lu and Vasil, 1981), Pennisetum americanum (Vasil and Vasil, 1981, 1982), P. purpureum (Vasil, Wang and Vasil, 1983) and Saccharum officinarum (Ho and Vasil, 1983). Such suspension cultures are currently the only source of gramineous protoplasts from which somatic embryos and plantlets can be obtained (Vasil and Vasil, 1980; Lu, Vasil and Vasil, 1981; Vasil et al., 1983; Vasil, 1983; Heyser, 1984; Srinivasan and Vasil, 1985). The present study describes morphological and ultrastructural changes occurring during the growth of embryogenic suspensions of Panicum maximum Jacq. (Guinea grass) and Pennisetum purpu' Received for publication 31 July 1985; revision accepted 20 November 1985. We thank Drs. Chin-yi Lu and Vimla Vasil for providing the embryogenic cell suspensions on which this work is based, and Drs. Henry C. Aldrich, and R. Howard Berg and Mr. Mark Taylor for assistance with electron microscopy. Supported by the Graduate School, Univ. of Florida, and a cooperative research project between the Gas Research Institute and the Institute of Food and Agricultural Sciences. Florida Agriculture Experiment Station Journal Series No. 6946. reum Schum. (Napier grass). A companion study describes the growth, cytology and flow cytometry of these suspensions (Karlsson and Vasil, 1986). MATERIALS AND METHODS-Initiation and maintenance of embryogenic suspensionsEmbryogenic cell suspension cultures used in this study were derived from embryogenic callus cultures obtained from immature inflorescences of Panicum maximum (for details see Lu and Vasil, 1981) and Pennisetum purpureum (for details see Vasil et al., 1983) in the spring of 1980 and summer of 1981, respectively. The suspensions were maintained in Murashige and Skoog's (1962) basal medium supplemented with 5% coconut water, 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 30 g/l sucrose for P. maximum, and with 5% coconut water, 2.5 mg/l 2,4-D, 0.2 mg/l thiamine hydrochloride and 30 g/l sucrose for P. purpureum. The pH was adjusted to 5.8 and the medium sterilized by autoclaving for 15 min at 15 psi and 121 C. The suspensions were subcultured routinely every 4-5 days by shaking the culture, letting it settle for a few seconds and then removing 2.5-3.0 ml of P. maximum and 8.0-9.0 ml of P. purpureum suspension from the top of the middle layer with an autopipette and transferring it to 35 ml of fresh medium in a 250