Embryo rescue has been used to produce maize-Tripsacum hybrids (Farquharson L.I., 1957. Heredity 48:295-299). Nevertheless the number of hybrids obtained could be increased through the induction of long-term embryogenic callus cultures from the rescued embryos (Furini A. and Jewell C., 1995. Maydica 40:205-210). The purpose of this work was to obtain long-term organogenic or embryogenic callus cultures to regenerate a high number of plants from maize-Tripsacum crosses. The genotypes used were Zea mays L. cv. Colorado Klein x cv. Ever Green (Zm20, 2n=20); inbred N107B (Zm40, 2n=40) from the Maize Genetics Coop. Stock Center, Urbana, Illinois, USA and Tripsacum dactyloides (2n=72).
Plants were grown in the greenhouse during the spring of 1995, 1996 and 1997. Crosses were made by removing the husk leaves and shortening the silks immediately before the pollen was applied (Mangelsdorf, 1974). The ears were sprayed with a solution of 40 mmolL-1 2,4-dichlorophenoxyacetic acid (2,4-D) to improve caryopsis development. They were harvested 12 to 14 days after pollination (dap), caryopses were disinfected with 2.5% sodium hypochlorite, embryos were excised and plated on culture medium and incubated at 28 C-30 C with a 16 h photoperiod. Embryos were cultured on García et al. basic medium (García et al., 1992. Revista de la Fac. de Agron. UNLP 68:15-25) supplemented with the following combinations of 2,4-D and 6-benzylaminopurine (BA)(in mmolL-1): 0-0 (A); 4-0 (B); 1,3-1,3 (C); 2,6-1,3 (D). Callus cultures were subcultured monthly in medium B. Shoots and somatic embryos were subcultured on medium A for plant regeneration. The chromosome number was determined in the root tips, which were pre-treated with 8-hydroxyquinoline and fixed in ethanol-acetic acid (3:1) solution and stained with ferric haematoxylin or Feulgen reactive.
The results differed according to the maize used as female parent:
Hybrid ZT46 (Zm20 x T. dactyloides) Caryopses were turgid and embryos were 0.5 mm or less (globular and transition stage) 12 dap. Table 1 observations were made 50 days after culture initiation. Organogenic callus induction was significantly higher in those media containing both plant growth regulators (2,4-D + BA), but plant regeneration and later transplanting were not efficient. Only 5 plants, 3 to 5 cm height with 1 to 3 adventitious roots, were regenerated but they all died during the transplanting period.
Nevertheless plants with normal root development were regenerated from callus cultures growing on medium with 2,4-D as the only growth regulator. The 6.25 % of the embryos plated on this medium gave rise to callus able to regenerate plants by organogenesis and somatic embryogenesis for a 12 month period. Eighteen plants are growing in the greenhouse. All of them showed a chromosome number of 2n = 46.
Hybrid ZT56 (ZM40 x T. dactyloides) Caryopses were turgid and embryos were 1.5 mm length (scutellar stage) at 12 to 14 dap. Embryogenic callus arose from 100% of the embryos and they regenerated plants during 24 months after plating. Some of the somatic embryos and organogenic callus were transferred to media A since the first month and up to date they gave rise to 188 vigorous plants with many adventitious roots, 77 of which are growing in the greenhouse. A chromosome number 2n=56 was determined in the regenerated plants.
Hybrid phenotype Both hybrids showed some similar vegetative
characteristics: they are tillering plants with Tripsacum-like leaves
and short rhizomes. Plant height ranged from 0.5 to 1 m in ZT46 and 1 to
2.9 m in ZT56. Hybrid ZT56 plants placed in the greenhouse in October 1997
started flowering by December. The lateral inflorescences were distich
ears, intermediate between maize and Tripsacum. The apical inflorescences
were tassels with 3 to 5 lateral branches. Although the pollen was sterile,
viable seeds were obtained from April 1998 to June and from September onwards.
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