Tissue cultures competent to regenerate plants have been isolated previously in this laboratory from three cv A188 tissue sources; the scutellum of immature embryos, the nodal stem sections of 14-day-old seedlings, and immature tassel flowers. An experimental approach combining a standard culture medium and routine incubation conditions with a specific visual selection process was successful in each case (Gordon, Robarts and Rice, MGNL 51:79-80, 1977; Rice, Reid and Gordon, Progagation of Higher Plants Through Tissue Culture, Univ. of Tenn. Symposium Proc., p. 262, 1979). The same approach has now been employed to establish competent, totipotent cultures from undeveloped axillary and secondary ears. The strategy for using our tissue culture methods to initiate totipotent cultures from a new meristematic tissue source was to focus on the characteristics of the explant. The variables involved included the age and physiological characteristics of the source plant, the developmental characteristics of the axillary and secondary ears, the nature of the explant itself and the orientation of the explant on the medium.
Greenhouse grown plants of cv A188 were used for this study. Axillary ears were taken from the lower nodes and secondary ears were removed from the shank of the primary ear. Axenic explants were successfully established from 49 ears. Totipotent cultures were established in 3 separate experiments from a total of 4 ears, 2 axillary ears and 2 secondary ears. These four ears were all less than 1 cm in length, and appeared morphologically normal, healthy and fully formed. They were removed from three plants which were 126, 135, and 137 days old respectively. In the successful experiments, the ears were sliced transversely, or sectored longitudinally and later sliced transversely, into thin tissue slices (< 0.1 cm); transverse slices were placed with basal side in contact with the culture medium. Within 3-4 weeks, some explants generated complex tissue/organ cultures containing totipotent tissues which were selectively excised and subcultured. The cultures have been maintained for five months and are morphologically indistinguishable from cultures derived from other tissue sources. Numerous plants have been regenerated and grown to maturity.
It is not clear based on these preliminary experiments whether positive results depend on using ears at a particular developmental stage, and the specific tissue from which cultures are initiated is not yet known. However, these results demonstrate the presence in mature plants of tissues which remain competent to establish totipotent cultures. (Supported in part by NSF Grant #75 20882 administered through Michigan State University).
S. J. Molnar, P. N. Gordon and T. B. Rice
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