We have succeeded in establishing liquid suspension cultures of A69Y opaque-2 endosperm cells (seeds from the collection of the Ist. Sper. Cereal., Sez. Bergamo). Two-year-old calli, induced from immature endosperms as described (Manzocchi et al., 1989) and cultivated in agar medium containing Murashige and Skoog salts, 30 g/l sucrose, 2 g/l asparagine and 1 mg/l thiamine (MSE), were transplanted frequently (every 15 days) for 5 subcultures to make them more friable, and were transferred to liquid MSE medium.
Actively growing suspensions were obtained from the smaller cell clusters, and separated from larger aggregates at each subculture; the tendency of cells to aggregate in clusters larger than 500 um is higher than in control cultures from A69Y+ endosperms.
Endosperm suspension cultures accumulate zein in lower amount, with respect to fresh weight and total protein, compared to developing endosperms (Manzocchi et al., Plant Cell Rep. 7:639-643, 1989); nevertheless, as opposed to o2 callus grown on solid media in which no zein protein could be detected, o2 liquid cultures synthesize detectable amounts of zeins, approximately one half of the amount accumulated in wildtype cells.
SDS electrophoretic patterns of ethanol-soluble pro-teins show, for o2 cells, the reduction of the heavier zein bands typical of the mutation.
Our data confirm, for the storage protein
mutations, the phenotypic expression of endosperm mutations in cultures,
as described for wx and ae by Saravitz and Boyer (Theor.
Appl. Gen. 73:489, 1987).
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