Universidad Nacional de Lomas de Zamora


Universidad Nacional de La Plata

Genotype and embryo age affect plant regeneration from maize/Tripsacum hybrids --García, MD, Molina, MdelC, Pesqueira, J The objective of this work was to evaluate the effects of genotype, embryo age and 2,4-Dichlorophenoxyacetic acid (2,4-D) concentrations on the induction of somatic embryogenesis and plant regeneration from maize/Tripsacum hybrids. Plant materials used for this experiment were Zea mays ssp. mays (2n=20): i) Inbred A188, from Dr. Phillips; ii) Inbred Santa Catalina 75 (SC75); iii) Cultivar Colorado Klein (CK) and Tripsacum dactyloides (2n=72), from Dr. Bird (CIMMYT). The hybrids obtained were i) Inbred A188 x T.dactyloides (A188xT); ii) Inbred SC75 x T. dactyloides (SC75 x T); iii) Cultivar Colorado Klein x T. dactyloides (CKxT); iv) Cultivar SCD x T. dactyloides (SCD x T).

Mother plants were grown in the greenhouse during 1998 spring and summer and crossings were practised during December. Maize ears were kept covered with paper bags until silk emergence. Silks were cut immediately before hand pollination with Tripsacum pollen and then ears were covered again with paper bags to prevent foreign pollen contamination. Ears were harvested 13, 15 or 19 days after pollination (dap). Developed caryopses were cut off and disinfected with 2,5% sodium hypochlorite solution. Embryos were isolated from the caryopsis, plated on culture media and incubated at 28-30 C with a 16 hour photoperiod. Culture media were composed of García et al. (Rev de la Fac de Agron de la UNLP 68:15-25) basic medium free of plant growth regulators (G0) or with the addition of different 2,4-D concentrations (µmol.L-1): 2.3 (G2) or 4.6 (G4). Calli were maintained on G4 medium and subcultured monthly. Shoots, obtained on the initiation media, were rooted on G0 medium under the same conditions of temperature and photoperiod. Regenerated plants were transplanted to pots with a plastic cover and three weeks later they were moved to the greenhouse. All the crossings performed gave rise to ears with developed caryopses only in the upper third. Caryopses were turgid with translucent endosperm until 15 dap. Embryo length ranged from 0.3 to 0.5 mm. Caryopses collapsed 19 dap because of endosperm development abnormalities, and by this time embryo length was very heterogeneous, from 0.5 to 2 mm. Only CKxT embryos showed a more uniform size, 1.5 to 2 mm.

Embryos plated on G0 medium germinated precociously (Table 1), but gave rise to weak plants without adventitious roots, which died during the rustication period.

Embryos grown on G4 medium showed low germination percentages but somatic embryogenesis was observed from all the hybrids (Table 2). Although frequencies of organogenic or embryogenic calli did not vary considerably amongst genotypes, the average of plants regenerated per responsive embryo during the culture period (8 months) was very different: 12.5 in A188xT, 2.5 in SC75xT, 2 in CKxT and 0.67 in SCDxT (Table 2).

On G2 medium, germination percentage has also been low and somatic embryogenesis frequencies were 16.6% for A188xT and 20.83% for SCDxT. These values did not differ significantly from those observed in G4 medium (c2 = 0.005; P£ 0.001).

Embryo age notoriously affected somatic embryogenesis frequencies. On G4 medium SC75xT embryos isolated 15 dap showed an induction frequency of 21.87% and 6.15% for those isolated 19 dap (c2 = 5.27; P£ 0.001). No plants were regenerated from calli obtained from embryos isolated 19 dap.

All regenerated plants showed a chromosome number 2n= 46.

In conclusion: Hybrid maize/Tripsacum embryos isolated 13 days after pollination and cultured on plant growth regulator free medium germinated but gave rise to weak plants that didnít survive. On the other hand, those embryos plated on medium supplemented with 4.6 µmol.L-1 2,4-D originated, by somatic embryogenesis and organogenesis, an average up to 12.5 vigorous plants/embryo.

The frequency of regenerated plants, of each genotype, was affected mainly by the callus regeneration ability more than the induction frequency of somatic embryogenesis.

Only the embryos less than 1 mm length (isolated 13 to 15 dap) originated calli able to regenerate plants by organogenesis or somatic embryogenesis.

Table 1: Germination frequencies of Maize/Tripsacum hybrid embryos isolated 13 dap and cultured on G0 medium. Observations were made 2 months after embryo plating.
Genotype Number of embryos Germination (%)
A188xT 21 52.4
SCDxT 24 79.2

Germination frequencies did not show significant differences among genotypes (c2 = 3.62; P£ 0.001).

Table 2: Somatic embryogenesis or organogenesis frequencies observed in maize/Tripsacum hybrid embryos isolated 13 or 15 dap and cultured on G4 medium. Observations were made 2 and 8 months after embryo plating.
Genotype Organogenesis or somatic embryogenesis frequency Number of regenerated plants Number of embryos
  2 mos1 8 mos2 2 mos 8 mos  
A188xT 25.00 11.11 9 50 36
SC75xT 21.87 12.50 4 10 32
CKxT 6.25 6.25 0 2 16
SCDxT 13.64 13.64 4 2 22

Embryogenesis frequencies between genotypes do not differ significantly: 1 c2 = 3.13; P£0.001; 2 c2 = 1.38; P£0.001.

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors.

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