Examination of the direct method of obtaining embryogenic cell suspensions of maize
--Piralov, GR, Abraimova, OE

The creation of cell selection technology requires reproducible methods of obtaining embryogenic cell suspensions for a wide range of genotypes. At present such cell suspensions of maize are obtained by the indirect method, based on the disintegration in liquid medium of friable embryogenic callus of type ll. In this report the results of examination of the direct method of obtaining cell suspensions of maize is described (Mezentsev A.V. et al., Dokl. VASHNIL, 9:3-5, 1987). Cultures were induced from seedling pieces of F1 hybrid Slavutich3 in N6 medium with L-asparagine (400 mg/l), L-proline (1200 mg/l), 2,4-D (4-6 mg/l) and were incubated in the dark at 27-28 C on a gyratory shaker (120 rpm). The process of cell suspension formation was going on in the 2 stages. In the first stage, which continued about 20 days, the heterogenous culture with single cells and groups (2-5 cells) was formed. The first cells were elongated as a rule, and were observed approximately at 5-6 days of growing, while the first oval to round small cells were found at 8-10 days. At 4-5 days of cultivation the liquid medium in vessels became very mucilagenous and therefore it was substituted every 3-4 days.

At day 20 of cultivation the density of cultures was about 1 million cells/ml. Their viability determined by the method of Widholm (Widholm J.M., Stain Technol., 47:189-194, 1972) was 50-60 %. The cultures were well dispersed and contained single cells and groups (2-10 cells). After 3 weeks, the old medium was discarded and cultures were transferred to new vessels. To these vessels 15 ml of fresh medium was added.

In the second stage the growth rate of suspensions increased and resulted predominantly in cultures of oval to round small cells with dense cytoplasm. We selected several sublines with a high rate of growth from the culture. The sublines were maintained by subculturing every 7 days on initial medium (5-7 ml inoculum on 15 ml fresh medium). Their growth subordinated to the exponential law with doubling time about 48-72 h in log-phase. In the cultures on the medium for differentiation (2,4-D - 0.1 mg/l) we observed somatic embryoids. Regeneration potential of these cultures is being researched now.
 

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