University of Paris

Effect of gelling agent on callus initiation from immature embryos of inbred A188 --V. Tremellat, P. Vain and P. Flament To obtain maize totipotent protoplasts, finely dispersed and embryogenic suspensions are to be used as starting material (Rhodes et al., Biotechnology 6:56-60, 1988). To establish such suspensions, friable and embryogenic calluses (referred to as type II calluses, as opposed to type I, which are embryogenic but compact) are needed. Unfortunately the ability to obtain type II callus is limited to a reduced number of genotypes (especially inbred A188) and remains difficult.

In order to enhance type II callus initiation, more favourable culture conditions have been researched. We have already shown that AgNO3, when added to a modified Murashige and Skoog's medium, increases 4 to 6 fold the rate of type II callus initiation from inbred A188 (Vain et al., Plant Cell Tissue Organ Cult., in press). Here, the effect of three classical gelling agents on maize callus initiation rate is reported.

Fourteen-day-old embryos (1-2 mm long) were aseptically removed from self-pollinated kernels of inbred A188 and plated on Murashige and Skoog's modified medium complemented with 10 mg/l AgNO3, with the embryo axis facing the medium. Cultivated embryos and/or calluses were subcultured every two weeks and callus production was scored at the fifth subculture. Results are given in the following table.

Gelling agent                                                                         Callus frequency
type I
type II
Gelrite (4g/l)
Agar (8g/l)
Agarose (6g/l)

Gelrite is from Siccap-Emmop (France), agar from Difco and agarose from Sigma (Type 1:low EEO); given concentrations are the most commonly used and give roughly the same compactness to the medium.

Both agarose and gelrite proved to be very well-suited for type I callus initiation (non-friable callus). For type II callus production, agarose gave the best results, showing a higher sensitivity of this type of callus to the impurities present in gelling agents. These results suggest that according to what is wanted and to the price of those components, using gelrite or agarose rather than agar may be worthwhile.

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