All of the experiments described in this communication were conducted with a field F1 generation of the intergeneric hybrid between Zea mays and Tripsacum dactyloides and its reciprocal crosses, obtained by Maria del Carmen Molina in our Institute.
In the hybrids, when T. dactyloides was used as female parent, the seeds are completely enclosed in a fruit case made up of a horny rachis and a hardened glume like Tripsacum. To overcome this barrier, the fruit cases were nicked with a scalpel and all seeds with a recognizable endosperm and well-visible signs of embryo development and health were selected for culture. Mature seeds were sterilized following the procedure of Green et al. (Crop Sci. 14:54-58, 1974). Embryos were aseptically excised from the seeds, but the residual endosperm was difficult to remove and several embryos were cultured with minor parts of endosperm attached to the embryo.
Callus was initiated and maintained on modified Murashige-Skoog culture medium (Green and Phillips, Crop Sci. 15:417-421, 1975). The medium was adjusted to pH 6.0 and then autoclaved for 20 min at 15 psi. The embryos and subsequent callus cultures were grown at 30/20 C and exposed to 12/12 hours photoperiod.
The embryos of the hybrid Z. mays X T. dactyloides and its reciprocal crosses were cultured for 30 days on medium containing 2,4-D at 0, 2, 5, 10, and 15 ppm. In the absence of 2,4-D the mature embryos, cultured with the scutellum side downward, germinated normally and after 21 days presented abundant roots and 2-3 green leaves. However, when these plantlets were transferred directly from the in vitro condition to the soil in the greenhouse they did not survive. An intermediate subculture in vermiculite is necessary to improve the survival rate.
In the presence of 2,4-D at any level, all T. dactyloides x Z. mays embryos cultured with the scutellum side upward failed to grow as callus. However, Z. mays x T. dactyloides embryos showed callus initiation dependent on the 2,4-D concentration used in the media (Fig. 1).
The growth response observed in the cultures was similar to that described by Cure and Mott (Physiol. Plant. 42:91-96, 1978) as yellowish compact, callus-like Class 3. The optimal growth rate was observed at 5 ppm 2,4-D. Each primary culture derived from 5 ppm 2,4-D medium produced 2 subcultures. After a 30 day cycle, each subculture produced 2 to 3 more subcultures. All subcultures were maintained in 2 ppm 2,4-D medium, but the 5 ppm 2.4-D derived cultures always showed the best growth. One of the cultures exhibited localized chlorophyll regions and one green 6 mm leaf from the callus.
When these tissues were excised and subcultured in media lacking 2,4-D to promote organ formation, none of them produced roots of any type, except the 5 ppm 2,4-D derived cultures surviving in medium without 2,4-D. Moreover, callus established and maintained with 5 ppm 2,4-D did not grow when transferred to a medium free of 2,4-D.
The lack of organ formation in such cultures could be related to Green's observation (Hortsci. 12:131-134, 1977) on the relation of embryo age-size and percent of callus capable of plant regeneration in maize.
In the next year we shall work with immature embryos of these hybrids.
Miguel Angel Rapela
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