Somatic embryogenesis and plant regeneration from callus cultures

Recently it has been demonstrated that cells of certain tissues of cereal and grass species can express totipotency via somatic embryogenesis (V. Vasil and I. K. Vasil, TAG 56:97, 1980). In 1982, C. Lu et al. (TAG 62:109, 1982) and C. E. Green (Proc. V Intern. Congr. Plant Cell Tissue Cult., Tokyo, Abstr., 1982) reported on somatic embryogenesis in Zea mays.

Immature embryos (1.1 - 1.5 mm in length) were taken from selfed plants of Chinese material Chi 31, a genotype appropriate to being used in maize tissue culture work (Y.-j. Xie and B. Gengenbach, MNL 55:95, 1981). Embryos were cultured with the scutellum facing upwards and the plumule and radicule sides in contact with agar. The MS and N-6 media (Chu et al., Sci. Sin. 18:659, 1975) were supplemented with 2.5 uM 2 , 4-D and 120 g/l sucrose (Lu et al.). The cultures were maintained at 25 C under 16-hour photoperiod. A compact, yellowish opaque callus was produced by proliferation of the scutellum one week after the initiation of the culture. The embryo axis was not involved in callus formation. Well-organized somatic embryos were formed on the surface of the scutellar callus within 2 to 3 weeks of the culture. The embryoids formed showed characteristic features (similar to zygotic maize embryos)--scutellum, coleoptile and coleorhiza. Numerous atypical embryoids were observed, however, especially those with two or more shoot apices. The scutellum of embryoids had a strong tendency toward secondary proliferation and embryogenesis. The germination of embryoids took place on the same basal media with 2.5 uM 2,4-D. The embryoids transferred singly or in clumps onto MS medium without 2,4-D but, containing 1 ppm GA3 (Lu et al.), germinated irregularly and did not form plants transplantable into soil. Well-developed plants with roots were formed on 2,4-D media. The regenerants were placed into perlite saturated with Hoagland's solutions, and 2 weeks later into potting soil. At present there is a total of 322 plants growing in a greenhouse. Regenerants originated from 90 initial explants and they were formed during 4 months of the culture. The callus is subcultured every 20 days and its embryogenetic capacity is maintained. All regenerated plants were green without any sign of albinism, but abnormal phenotypes occurred in the seedlings: a prostrate growth habit, tillering, leaves with white or yellow stripes, virescent types. Regenerated plants show variability in growth. Chromosomal analysis, and scoring for the presence of mutation in the progenies derived from regenerated plants are in progress.

F. J. Novak, M. Dolezelova, M. Nesticky, A. Piovarci

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