In the aim of developing cell suspension cultures maintaining morphogenic competence, with a potential use in obtaining protoplasts capable of plant regeneration (K.K. Kamo et al., Planta 172:245, 1987), we are studying the conditions to grow liquid cultures from callus derived from genetic stocks with good embryogenic potential (A188, W64A, Mo17 x B73, A188 x B73, W64A x A188).
Seeds were a kind gift from the Istituto Sperimentale per la Cerealicoltura, Bergamo, and the Dipartimento di Genetica e di Biologia dei Microorganismi, University of Milano. Plant-regenerating callus cultures were initiated both from immature embryos (1mm long) and from the nodal region of mature embryos separated from ripe seeds, on agar-containing MSE medium (MS salts + 25mM proline + 20g/l sucrose + 1mg/l 2,4-D), modified from Armstrong and Green (Planta 164:207, 1985). After subculturing every 2 weeks on the same medium for 2 months, the faster growing and more friable calli were transferred (1g fresh weight/30ml) to liquid MSE medium and incubated at 27 C on a rotary shaker. The medium was changed every week; cell release was evaluated after 1 month.
No appreciable release of cells was observed from morphogenic calli derived from mature embryos, which presented a compact structure which was not affected by different additions of 2,4-D to the culture medium; calli from immature embryos from all the genotypes examined, on the contrary, easily released cells which could be cultivated. The influence of 2,4-D on cell release was examined in detail on the two inbred lines: while for A188 increasing concentrations of the auxin (1, 1.5, 3mg/l) appear to increase cell release to liquid medium (from 0.9 to 1.5ml packed cells per 1g callus), for W64A the effect is reduced (the stimulation was from 0.8 to 1ml packed cells/culture).
Growth curves of cells in suspension cultures were determined on 15ml cultures, containing at the beginning 1ml packed cells: the growth was not very enhanced (2 fold increase after 1 week) and the plateau was reached after 2 weeks. Experiments are in progress to optimize conditions for cell release and suspension growth; the concentration of 2,4-D seems not to affect either the cell growth or the secretion of a thick white substance, which has been described to be secreted by maize liquid cultures (Kamo et al., 1987; J.A. Miernyk, J. Plant Physiol. 129:19, 1987).
Suspension cultures from embryogenic callus of all the genotypes examined are composed of small, round-shaped cells, which tend to aggregate in small clusters; these features are the same as those described by Kamo and Hodges (Plant Sci. 45:111, 1986) for embryogenic maize suspensions, and are characteristically different from the aspect of non-regenerating suspensions from root callus (single big elongated cells) obtained from the same genotypes.
Silvana Castelli and Lucia A. Manzocchi
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